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. 2016 Jun 16:6:27998.
doi: 10.1038/srep27998.

Rhizobial gibberellin negatively regulates host nodule number

Yohei Tatsukami  1   2 , Mitsuyoshi Ueda  1
Affiliations

Rhizobial gibberellin negatively regulates host nodule number

Yohei Tatsukami et al. Sci Rep. .

Abstract

In legume-rhizobia symbiosis, the nodule number is controlled to ensure optimal growth of the host. In Lotus japonicus, the nodule number has been considered to be tightly regulated by host-derived phytohormones and glycopeptides. However, we have discovered a symbiont-derived phytohormonal regulation of nodule number in Mesorhizobium loti. In this study, we found that M. loti synthesized gibberellic acid (GA) under symbiosis. Hosts inoculated with a GA-synthesis-deficient M. loti mutant formed more nodules than those inoculated with the wild-type form at four weeks post inoculation, indicating that GA from already-incorporated rhizobia prevents new nodule formation. Interestingly, the genes for GA synthesis are only found in rhizobial species that inhabit determinate nodules. Our findings suggest that the already-incorporated rhizobia perform GA-associated negative regulation of nodule number to prevent delayed infection by other rhizobia.

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Figures

Figure 1
Figure 1. GA-synthetic operon in M. loti and time-course plant assays.
(a) The operon consists of 9 genes with accession numbers mlr6364–mlr6372. The arrowhead indicates the transposon insertion site in the gib− mutant. (b) Flowering period from the day of inoculation. (c) Shoot length of the host plant when flowering. (d) Number of rhizobia grown in roots. (e) Nitrogen fixation activity determined by acetylene reduction assay. (f) Number of nodules. (g) Weight per nodule at 5WPI. Bars indicate the average weights of 30 nodules. (h) Relative frequency of nodule position from root-stem boundary at 8WPI. Significance was determined by Mann-Whitney U-test (p-value = 5.52 × 10−13). Relative frequencies were calculated by 210 for wild-type and 245 nodules for gib− mutant, respectively. (i,j) Nodules formed in root of L. japonicus inoculated with M. loti wild-type and gib− mutant at 8WPI. Red arrows indicate the nodules. Significances were determined by student t-test (b,c,g). Multiple comparisons were corrected using the Holm–Bonferroni t-test. *p < 0.05, **p < 0.01, ***p < 0.001 (d,e,f). WPI, weeks post inoculation. Error bars indicate SEMs from 3 plants (d), 8 plants (e), >15 plants (b,c,f) and 3 biological replicates (g).
Figure 2
Figure 2. Construction of gib+ mutant and GA identification by LC–MS analysis.
(a) Plasmid vector for homologous recombination of the M. loti genome. Lac promoter cassette in front of the homologous region (600 bp) is inserted into EcoRI restriction site. (b) Lac promoter insertion site. Note that gib+ mutant is a single-crossover mutant. (c) Relative expression levels of the operon genes. The fold-change values are relative to wild-type grown in liquid culture with 1 mM isopropyl β-D-1-thiogaractopyranoside (expression level = 1). Error bars indicate the standard error from 3 independent experiments. (d,e) NanoLC–MS chromatograms of culture medium of wild-type and gib+ mutant and authentic GAs. The chromatograms of mass range at m/z = 315.155–315.165 (d) and m/z = 345.165–345.175 (e) are extracted to focus on GA9 and GA24. (f–i) Mass spectra of gib+ culture medium at 56.5 min (f) and 53.7 min (g) and authentic GA9 (h) and GA24 (i). Insets show the structure of GA9 and GA24.
Figure 3
Figure 3. GA synthesis mechanism in nodules.
(a) GA-synthetic pathway in nodules. Reactions mediated by enzymes encoded on the GA-synthetic operon are shown inside the broken-lined box. (b) The amount of GA1 and GA3 in plants inoculated with wild-type and gib− mutant rhizobia were determined by LC–MS. GA4 and GA7 were below the detection limit under both conditions. Significant differences were determined using student t-test. *p < 0.05. Error bars indicate SEMs from 3 biological replicates. (c) Number of nodules. Water-dissolved GA3 was exogenously added at 10−7 mole per Incu Tissue at 2WPI. Multiple comparisons were corrected using the Holm-Bonferroni t-test. ***p < 0.001. WPI, weeks post inoculation. Error bars indicate SEMs from 9 plants.
Figure 4
Figure 4. Phylogenies of legumes and bacteria, nodule types and distribution of GA synthetic genes.
(a) Phylogenetic trees are modified from Doyle. The bacterial phylogeny (left) is based on the 16S rRNA gene sequence. Representative symbiotic bacteria are shown; lineages of non-symbiotic bacteria are indicated by boxes. The bacteria possessing putative GA synthetic genes are shown in red bold characters and the bacteria without are shown in blue bold characters. Whether a bacterial species has GA-synthetic genes was determined by the presence of both ent-copalyl pyrophosphate synthase and ent-kaurene synthase by BLAST search. The phylogenetic relationships of legumes (and some non-legumes) based on rbcL sequence (right) are indicated for selected legume genera nodulated by bacteria that are shown on the bacterial tree. Nodule type and taxonomy are shown to the right of the tree. Arrows connect bacterial symbionts with their plant host. The wide host-range of NGR234 is shown by its ability to nodulate Parasponia. (b) Nodule structure of determinate and indeterminate nodules. Rhizobia lose the ability to reproduce after differentiation into bacteroids in indeterminate but not determinate nodules. Rhizobia typically possess the gibberellin operon only in hosts with determinate nodules.
Figure 5
Figure 5. Competition between M. loti WT and gib−.
(a) Number of nodules in co-inoculation assays. Nodule numbers for gib− are shown in grey, taken from Fig. 1f. Error bars represent SEMs from > 8 plants. (b) The number and percentage of rhizobia in roots. The number above each bar indicates the percentage of gib− colonies compared with the total colonies. Error bars indicate the SEMs from 3 plants. †: at this point, bacteria are isolated from soil (1 g) immediately after co-inoculation of WT and gib−. (c) Co-cultivation assay in liquid medium. Error bars indicate the SEMs from 3 independent experiments.
Figure 6
Figure 6. Model of nodule number regulation by rhizobial gibberellin.
In the early developmental stage (no mature nodules), rhizobia inoculate into host plants only under host-derived control (e.g. auto-regulation of nodulation). After nodules have matured, the symbiont-derived regulation via rhizobial gibberellin also works to inhibit delayed infection by other rhizobia.
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