Rhizobium leguminosarum bv. trifolii NodD2 Enhances Competitive Nodule Colonization in the Clover-Rhizobium Symbiosis

Abstract

Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here, we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii contain a second copy designated nodD2. nodD2 genes were present in 8 out of 13 strains of R. leguminosarum bv. trifolii, but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in R. leguminosarum bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual R. leguminosarum bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained nodD2, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts.IMPORTANCE Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of nodD, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here, we identified and characterized a second copy of nodD present in some strains of the clover microsymbiont Rhizobium leguminosarum bv. trifolii. The second nodD gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing nodD2 could be utilized as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.

Keywords: Nod factor; NodD; Rhizobium leguminosarum; clover; competition; symbiosis.

FIG 1

Nodulation kinetics of white clover plants inoculated with R. leguminosarum bv. trifolii strain TA1 or its nodD mutant derivatives. Three biological replicates were performed with 10 technical replicates in each. (A) Average number of nodules per plant. (B) Percentage of plants nodulated. Error bars represent ± standard error of the mean (SEM). Asterisks represent significant difference from TA1 ΔnodD1 determined using one-way analysis of variance (ANOVA) performed on data from day 35 with Dunnett’s multiple comparisons post hoc test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The same statistical analysis was performed separately on data from day 11 for early infection.

FIG 2

Role of NodD1 and NodD2 in activation of nodA and nodF promoters. β-galactosidase assays were conducted on TA1 nodD mutant derivatives containing nodA or nodF promoter reporter plasmids in response to 10 μM synthetic DHF. The DMSO-only controls are superimposed on the DHF-induced treatment data. (A) nodA promoter induction. (B) nodF promoter induction. Each symbol represents a biological replicate. Error bars represent ± SEM. Asterisks indicate significant difference between the treated samples determined using one-way ANOVA (Tukey’s multiple comparisons post hoc test, *, P < 0.05; ***, P < 0.001; ****, P < 0.0001).

FIG 3

Infection thread formation on white clover by strain TA1 and its nodD mutant derivatives containing the reporter plasmid pPR3. The number of ITs per strain was observed at seven dpi on 10 plants, each represented by a symbol. The error bars represent ± the SEM. The asterisks indicate significant difference from the wild type (one-way ANOVA with Dunnett’s multiple comparisons post hoc test, **, P < 0.01).

FIG 4

The relative competitive ability of TA1 wild type and nodD mutant strains. The percentage of nodules occupied by each strain was determined by the presence of blue/nonblue nodules following staining of white clover roots inoculated with pairs of strains in a 1:1 ratio (approximately 500 cells of each strain). The proportion of blue nodules from each comparison is reported as percentage. The same pairs of strains containing reciprocal reporter plasmids are linked by bar color. In each reciprocal combination, the strain lacking pAMNHCELB contained the plasmid pAMNHGUSA. The percentage indicates the proportion of blue nodules determined from the sum of the five uppermost nodules formed on 10 plants (50 nodules total where 5 nodules were present). Three to four sets of 10 plants per strain pair were examined, with each replicate set represented by a symbol. The error bars represent ± the SEM. The asterisks indicate significant difference between the reciprocal pairs of strains (one-way ANOVA with Sidak’s multiple comparisons post hoc test, ***, P < 0.001; ****, P < 0.0001).

FIG 5

Alignment of TA1 NodD1 and NodD2 amino acid sequences and corresponding predicted protein structures shown as ribbon models constructed using Phyre2. (A) Amino acid sequence alignment. The background tone indicates amino acid similarity, where black is identical, dark gray is similar, light gray is not similar. The green bar indicates conserved residues. Three regions corresponding to the predicted flavonoid inducer binding pocket entrance are underlined in purple, blue, and red. (B) TA1 NodD1. (C) TA1 NodD2. The location of the residues highlighted in (A) are shown on the ribbon structures with corresponding colors.

FIG 6

Evolutionary history of 21 nodD nucleotide sequences inferred by using the maximum likelihood method and Tamura-Nei model (76). For strains with one copy, nodD1 is black; for strains with both, nodD1 and nodD2 are blue and green, respectively. The phenological and geographical distributions of the strains are indicated according to the key, based on information in Table 1. The tree with the highest log likelihood (−6,198.64) is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (77). Initial tree(s) for the heuristic search were obtained automatically by applying neighbor joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 21 nucleotide sequences. There were a total of 986 positions in the final data set. Evolutionary analyses were conducted in MEGA X (78).