Lifestyle adaptations of Rhizobium from rhizosphere to symbiosis

Abstract

By analyzing successive lifestyle stages of a model Rhizobium-legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N₂-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N₂ Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N₂ fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium-legume symbiosis.

Keywords: N2 fixation; Rhizobium; legume; nodulation; rhizosphere.

Fig. 1.

Collection steps following inoculation of pea plants with the INSeq input libraries. Bacterial DNA was purified from 1) the rhizosphere (5 dpi) and 2) colonized roots (5 dpi) following inoculation with input1; and from 3) nodule bacteria (28 dpi) and 4) bacteroids (28 dpi) following inoculation with input2. These four samples, together with DNA purified from input1 and input2, were used to make libraries for INSeq analysis.

Fig. 2.

Rlv3841 genes required during the stages of symbiosis with pea. Orange boxes show the number at each symbiotic stage: rhizosphere (17 genes) (SI Appendix, Table S1), rhizosphere-progressive (146 genes) (SI Appendix, Table S2), rhizosphere and root (7 genes) (SI Appendix, Table S3), root (23 genes) (SI Appendix, Table S4), root-progressive (33 genes) (SI Appendix, Table S5), nodule-general (211 genes) (SI Appendix, Table S6), nodule bacteria (142 genes) (SI Appendix, Table S7), and bacteroid (24 genes) (SI Appendix, Table S8). All genes shown were classified as NE in the respective input library.

Fig. 3.

Classification of genes encoding motility and chemotaxis proteins in rhizosphere, root, nodule bacteria, and bacteroid INSeq libraries. Genes required at a given stage are shown in red; genes where mutation has no effect (NE) are shown in white; genes that, when mutated, are advantageous are in green; and those unclassified due to too few TA sites are in black. (INSeq results are shown in SI Appendix, Table S10.) The proteins are colored by functional group, but the color is arbitrary with respect to INSeq phenotype.

Fig. 4.

Experimental characterization of mutant phenotypes predicted from INSeq data. (A) Root colonization by bacteria (cfu/g root) from a single-strain inoculation (10⁵ cfu) of a 7-d-old pea plant comparing wild type and a mutant in pRL100053 (classified as root-progressive) at 5 dpi (n ≥ 8). An unpaired t test was used to assess statistical significance. (B) Root colonization by bacteria (cfu/root) from a single-strain inoculation (10⁵ cfu) of a 7-d-old pea plant comparing wild type and a mutant in pRL80032 (root-progressive) at 5 dpi (n ≥ 4). An unpaired t test was used to assess statistical significance. (C) Nodule formation on peas (nodules/plant as a percentage of wild type [Rlv3841GusA, 180 ±19.2 nodules per plant]) from a single-strain inoculation (10⁴ cfu) for mutants in nodule-general genes (9), nodule bacteria genes (3), and a bacteroid gene (Table 1) at 28 dpi (n ≥ 3). A Dunnett’s test comparing each mutant to wild type was used to assess statistical significance. (D) Acetylene reduction as a percentage of wild type (Rlv3841GusA, 5.18 ± 0.380 μmol of ethylene per plant per hour) of mutants in nodule-general genes (9), nodule bacteria genes (3), and a bacteroid gene (Table 1) at 28 dpi (n ≥ 3). A Dunnett’s test comparing each mutant to wild type was used to assess statistical significance. (E) Competition for nodule occupancy of mutants with wild type (nodule percentage/inoculum percentage) from 1:1 (mutant:wild type) coinoculation (total, 10⁴ cfu) of pea plants with eight nodule-general mutants harvested at 21 dpi (n ≥ 5). Statistical significance was assessed by a mixed-effects model comparing the proportion of each strain in inoculum and nodules. (F) Bacterial recovery (cfu/mL [×10⁸]) from crushed nodules (10 per plant) and 36 h growth in TY broth at 28 °C for wild type and three nodule bacteria mutants (n ≥ 3). To assess statistical significance, a Dunnett’s test comparing each mutant strain to wild type was used. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Error bars show ±SEM.

Fig. 5.

Rlv3841 genes required at different stages of symbiosis with pea from INSeq analysis. Three environments (rhizosphere, root, and nodule) are shown. Progressive genes alter competition at subsequent stages. Gene requirements at each stage are discussed in Results, and full gene lists can be found in SI Appendix, Tables S1–S8.