1. Evolutionary Biology
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Spatio-temporal control of mutualism in legumes helps spread symbiotic nitrogen fixation

  1. Benoit Daubech
  2. Philippe Remigi
  3. Ginaini Doin de Moura
  4. Marta Marchetti
  5. Cécile Pouzet
  6. Marie-Christine Auriac
  7. Chaitanya S Gokhale
  8. Catherine Masson-Boivin  Is a corresponding author
  9. Delphine Capela
  1. Université de Toulouse, INRA, CNRS, France
  2. Massey University, New Zealand
  3. Fédération de Recherches Agrobiosciences, Interactions et Biodiversité, Plateforme d’Imagerie TRI, CNRS - UPS, France
  4. Max Planck Institute for Evolutionary Biology, Germany
Research Article
Cite this article as: eLife 2017;6:e28683 doi: 10.7554/eLife.28683
10 figures, 2 tables and 3 additional files

Figures

Figure 1 with 1 supplement
Kinetics of reproductive fitness of Fix+ and Fix- bacteria in nodules following co-inoculation of M. pudica.

M. pudica plants were co-inoculated with a mixture of Fix+ and Fix- strains at a 1/1 ratio, using 106 (A) or 1010 bacteria/plant (B). Nodules were individually analyzed by plating their bacterial population (see Figure 1—figure supplement 1). Co-infected nodules represented ca. 3% (A) or 20% (B) of the nodules. (A) The ratio of the mean number of bacteria per Fix+-containing nodule to the mean number of bacteria per Fix-- containing nodule was calculated for each individual plant at each time point (see Figure 1—figure supplement 1) and box plots represent the distribution of these ratios (Figure 1—source data 1). Only single-infected nodules were taken into account in this graph. (B) Box plots represent the distribution of the ratios of Fix+ bacteria to Fix- bacteria in co-infected nodules (Figure 1—source data 2). Central rectangles span the first quartile to the third quartile (that is, the interquartile range or IQR), bold segments inside rectangles show the median, unfilled circles indicate suspected outliers, whiskers above and below the box show either the locations of the minimum and maximum in the absence of suspected outlying data or 1.5 × IQR if an outlier is present. Horizontal dashed lines correspond to ratios equal to 1. The number of plants (A) or nodules (B) analyzed is indicated in brackets. *Significant differences between the number of Fix+ and Fix- bacteria per nodule (p<0.05, multiple comparison test after Kruskal-Wallis (A); p<0.001, after Student t-test with paired data (B).

https://doi.org/10.7554/eLife.28683.003
Figure 1—source data 1

Reproductive fitness of nodule bacteria following co-inoculation with Fix+ (CBM2700) and Fix- (CBM2707) C. taiwanensis.

https://doi.org/10.7554/eLife.28683.005
Figure 1—source data 2

Reproductive fitness of nodule bacteria in nodules co-infected by Fix+ (CBM2700) and Fix- (CBM2707) C. taiwanensis.

https://doi.org/10.7554/eLife.28683.006
Figure 1—figure supplement 1
Kinetics of reproductive fitness of nodule bacteria following co-inoculation with Fix+ (CBM2700) and Fix- (CBM2707) C. taiwanensis.

Strains were inoculated in ratio 1/1. M. pudica nodules were individually analyzed. Box plots were constructed as described in Figure 1. All nodules from each plant were analyzed individually (Figure 1—source data 1). The number of nodules analyzed is indicated in brackets.

https://doi.org/10.7554/eLife.28683.004
Kinetics of reproductive fitness of Fix+ or Fix- nodule bacteria following single-inoculation of M. pudica.

(A) Fix+ (CBM382) or Fix- (CBM2568) C. taiwanensis were inoculated on M. pudica. Box plots represent the distribution of the number of bacteria recovered per nodule on plates. Box plots were constructed as described in Figure 1. R, ratios of the median number of Fix+ bacteria per nodule on the median number of Fix- bacteria per nodule. The number of nodules analyzed at each time point is indicated in brackets. The number of plants analyzed at each time point is indicated in red. Results are from two independent experiments (Figure 2—source data 1). *Significantly different from the number of Fix+ bacteria per nodule (p<0.05 multiple comparison test after Kruskal-Wallis). (BC) Theoretical reproductive fitness of Fix+ (B) and Fix- bacteria (C) following single-inoculation of M. pudica as compared to experimental data. Dotted lines represent bacterial populations per plant averaged over 200 replicate simulations (Figure 2—source data 2). Box plots represent the distribution of the number of bacteria experimentally recovered per plant. Experimental data are from (A).

https://doi.org/10.7554/eLife.28683.007
Figure 2—source data 1

Reproductive fitness of nodule bacteria following single-inoculations with either Fix+ (CBM382) or Fix- (CBM2568) C. taiwanensis.

https://doi.org/10.7554/eLife.28683.008
Figure 2—source data 2

Simulation data for the reproductive fitness of Fix+ and Fix- bacteria following single inoculations of M. pudica.

https://doi.org/10.7554/eLife.28683.009
Relative number of nodules formed by Fix+ and Fix- bacteria per plant individual.

M. pudica plants were co-inoculated with the CBM2700 (Fix+, GFP) and CBM2707 (Fix-, mCherry) strains at a 1/1 ratio. The number of plants analyzed for each time point is indicated in brackets. Boxplots were constructed as described in Figure 1. No significant differences were observed between the number of nodules formed by Fix+ bacteria and Fix- bacteria per plant at the different time points (p>0.05, Student t-test with paired data at each time point or multiple comparison test after Kruskal-Wallis on the whole dataset) (Figure 3—source data 1).

https://doi.org/10.7554/eLife.28683.010
Figure 3—source data 1

Relative number of nodules formed by Fix+ and Fix- bacteria per plant individual.

https://doi.org/10.7554/eLife.28683.011
Nodulation kinetics.

M. pudica plants were single-inoculated with either CBM832 (Fix+) or CBM2568 (Fix-) or co-inoculated with a mixture of both strains at a 1/1 or 1/99 ratio. First nodules appeared at 5–7 dpi (Figure 4—source data 1).

https://doi.org/10.7554/eLife.28683.012
Figure 4—source data 1

Nodulation kinetics of Fix+ (CBM382) and Fix- (CBM2568) C. taiwanensis following single- or co-inoculation of M. pudica.

https://doi.org/10.7554/eLife.28683.013
Viability of Fix+ and Fix- bacteroids.

M. pudica were co-inoculated with Fix+ and Fix- C. taiwanensis at a 1/1 ratio and sections of nodules collected at 14 dpi (ABFG), 16 dpi (C) or 35 dpi (DEHI) were observed under bright field (panels 1) or fluorescent microscopy (panels 2 and 3), and after PI staining (panels with an *). Panels with the same letters represent the same nodule section. (F3), magnification of (F2) visualized by confocal microscopy. (A) and (D), sections of nodules infected with a GFP-labeled Fix+ strain. (B) (C) and (E), sections of nodule infected with a GFP-labeled Fix- strain. (F), nodule co-infected with a GFP-labeled Fix+ and a mCherry-labeled Fix- strain. (G) and (H), nodules co-infected with a GFP-labeled Fix- and an unlabeled Fix+ strain. (I), nodules co-infected with a GFP-labeled Fix+ and an unlabeled Fix- strain. The white and yellow dotted lines in (GHI) delimit the areas occupied by the Fix- and Fix+ strains in a co-infected nodule, respectively. Note that neither the Fix+ (D3) nor the Fix- bacteroids (B3G3) are red-labeled by PI staining at 14 dpi whereas a few cells are PI-stained in the Fix--occupied nodule at 16 dpi ([C3], arrows), and Fix- are mostly PI-labeled (dead) at 35 dpi (E3H3I3). Note that bacteria of the infection zone are still alive at 35 dpi (arrow, E2E3). Note that nodule cells filled with Fix- are browner than nodule cells filled with Fix+ (G1H1I1). Scale bars correspond to 100 µm except for F3 (30 µm).

https://doi.org/10.7554/eLife.28683.014
Electron microscopy of Fix+- and Fix--occupied nodules.

M. pudica plants were co-inoculated with Fix+ (CBM2708, mCherry) and Fix- (CBM2568, unlabeled) C. taiwanensis at a 1/1 ratio. Nodules collected at 19 dpi (ABCDE) were sorted for mCherry expression under fluorescence microscopy and used for electron microscopy observation. Degenerated nodule cells (*) were observed in Fix--occupied nodules (BDE) but not in Fix+-occupied nodules (AC). (C) and (D) represent magnification of the zones delimitated by a black dashed rectangle in (A) and (B) respectively. (E) magnification of the white rectangle in (D) showing degenerated bacteria (arrows). Scale bars represent 20 µm (ABC), 10 µm (D) and 2 µm (E).

https://doi.org/10.7554/eLife.28683.015
Kinetics of nitrogenase activity in N2-fixing M. pudica nodules.

Plants were inoculated with C. taiwanensis CBM832 (Fix+), and nitrogenase activity measured using the acetylene reduction assay (ARA) (Figure 7—source data 1). Two negative controls, i.e. tubes containing only the acetylene substrate and plants inoculated with C. taiwanensis CBM2568 (Fix-), were included. In these cases, boxplots correspond to data from all time points. *, Significantly different from the negative controls (p<0.05 after multiple comparison test of Kruskal-Wallis).

https://doi.org/10.7554/eLife.28683.016
Figure 7—source data 1

Nitrogenase activity of C. taiwanensis Fix+ (CBM832).

https://doi.org/10.7554/eLife.28683.017
Experimental and theoretical reproductive fitness of Fix+ and Fix- bacteria following co- inoculation of M. pudica (ratio 1/100).

The proportion of Fix+ clones in nodules was experimentally measured and simulated over 49 days, following co-inoculation of 20 plants. Experimental data are shown as black triangles (Figure 8—source data 1). Black error bars represent standard deviation from 2 to 3 replicates. The results from 100 replicate simulations are shown as grey dots and boxplots (Figure 8—source data 2).

https://doi.org/10.7554/eLife.28683.018
Figure 8—source data 1

Experimental data for the reproductive fitness of Fix+ and Fix- bacteria following co- inoculation of M. pudica (ratio 1/100) over 49 days.

https://doi.org/10.7554/eLife.28683.019
Figure 8—source data 2

Simulation data for the reproductive fitness of Fix+ and Fix- bacteria following co- inoculation of M. pudica (ratio 1/100) over 49 days.

https://doi.org/10.7554/eLife.28683.020
Figure 9 with 1 supplement
Effect of cycle length and plant numbers on the predicted distributions of Fix+population sizes.

Model simulations were performed with an initial proportion of 1% Fix+ in the bacterial population inoculated to a pool of plants. The length of each cycle and the number of plants per pool varied as indicated in the legend. (A) Final proportion of Fix+ clones after four cycles (Figure 9—source data 1). Boxplots represent the distribution of the final proportion of Fix+ clones from 100 simulations. The length of inoculation cycles ranged from 14 to 49 days and the number of plants per pool from 1 to 100. Numbers underneath each boxplot indicate the number of replicate simulations where Fix+ sub-populations became extinct after four cycles. (B) Increase in the proportion of Fix+ clones along 10 inoculation cycles of 14, 21, 28, 35, 42 or 49 days (Figure 9—source data 2). The number of plants per pool was 20. Representative trajectories of 5 replicate pools are shown in each case.

https://doi.org/10.7554/eLife.28683.022
Figure 9—source data 1

Simulation data for the final proportion of Fix+ bacteria after four inoculation cycles.

https://doi.org/10.7554/eLife.28683.024
Figure 9—source data 2

Simulation data for the increase in proportion of Fix+ bacteria along 10 cycles.

https://doi.org/10.7554/eLife.28683.025
Figure 9—source data 3

Simulation data for the effect of cycle length and plant number on the Fix+population sizes after four cycles.

https://doi.org/10.7554/eLife.28683.026
Figure 9—figure supplement 1
Effect of cycle length and plant numbers on the predicted distribution of Fix+ population sizes.

Model simulations were performed for four cycles with an initial proportion of 0.1% Fix+ in the bacterial population inoculated to a pool of plants of sizes ranging from 1 to 1000 plants. The length of each cycle and the number of plants per pool varied as indicated in the legend. Numbers underneath each boxplot indicate the number of replicates (out of 100) where Fix+ sub-populations became extinct after four cycles (Figure 9—source data 3).

https://doi.org/10.7554/eLife.28683.023
Frequency of Fix+ bacteria over 4 cycles of 35 (A) or 21 (B) days: simulations and experimental validation.

The proportion of Fix+ clones over four inoculation cycles was simulated and measured experimentally. Simulations and experiment were performed with an initial proportion of Fix+ clones of 1% and pools of 20 plants. Experiments were performed with an inoculum of 5 × 103 Fix+/5 × 105 Fix- C. taiwanensis per plant. The results from 100 replicate simulations are shown as grey dots and boxplots (Figure 10—source data 1). Experimental data are shown as black triangles (Figure 10—source data 2).

https://doi.org/10.7554/eLife.28683.027
Figure 10—source data 1

Simulation data for the frequency of Fix+ bacteria over 4 cycles of 35 or 21 days.

https://doi.org/10.7554/eLife.28683.028
Figure 10—source data 2

Experimental data for the frequency of Fix+ and Fix- bacteria over 4 cycles of 35 or 21 days.

https://doi.org/10.7554/eLife.28683.029

Tables

Table 1
Model parameters
https://doi.org/10.7554/eLife.28683.021
ParameterAbbreviationValue
Size of each pool of plants*PoolVariable (1–1000)
Number of replicates*RepVariable (5 or 100)
Length of each cycle*DaysVariable (14-49)
Number of cycles*CycVariable (4 or 10)
Initial proportion of Fix+ cells*xVariable (1 or 0.1)
Maximum number of new nodules/plant/dayλmax0.44
Coefficient for the auto-regulation of nodulation in nodulation kineticsa10.03
Coefficient for time-decay in nodulation kineticsa20.006
Lag for time-decay in nodulation kineticsa32
Growth rate of bacteria within noduler1.95
Fitness cost of nitrogen fixation c0
Sanctions for Fix-‡s1.65
Day at which additional sanctions beginds17
Nodule carrying capacityK1.4 × 108
  1. *parameters varied in the simulations

    experimentally measured parameters

  2. parameters inferred from experimental data

Table 2
Strains and plasmids used in this study
https://doi.org/10.7554/eLife.28683.030
BacteriumStrainRelevant characteristicsReference/source
E. coliDH5αF recA lacZM15Bethesda research laboratory
DH5α λpirF recA lacZM15 λpirHP Schweizer
C. taiwanensisLMG19424Wild-type strain isolated from Mimosa pudica in Taiwan(Chen et al., 2001)
CBM832LMG19424 derivative resistant to Streptomycin, StrRM. Hynes
CBM2568CBM832 deleted in nifH, StrRThis study
CBM2700CBM832 carrying a Pps-GFP fusion downstream glmS, StrRThis study
CBM2701CBM2568 carrying a Pps-GFP fusion downstream glmS, StrRThis study
CBM2707CBM2568 carrying a Pps-mCherry fusion downstream glmS, StrRThis study
CBM2708CBM832 carrying a Pps-mCherry fusion downstream glmS, StrRThis study
PlasmidsNameRelevant characteristicsReference/source
pGPI-SceIoriR6K, mob+, carries a I-SceI site, TriR(Flannagan et al., 2008)
pDAI-SceIoripBBR1, mob+, carries the I-SceI gene, TetR(Flannagan et al., 2008)
pRCK-Pps-GFPPlasmid carrying the psbA promoter region fused to GFP, KanRM. Valls
pRCK-Pps-mCherryPlasmid carrying the psbA promoter region fused to mCherry, KanRM. Valls
pCBM156pGPI-SceI carrying the nifH 5’ and 3’ regions, TriRThis study
pCBM161pGPI-SceI carrying the glmS-Ralta_A0206 intergenic region interrupted by a Pps-GFP fusion, TriRThis study
pCBM162pGPI-SceI carrying the glmS-Ralta_A0206 intergenic region interrupted by a Pps-mCherry fusion, TriRThis study
pRK2013Helper plasmid, KanR(Figurski and Helinski, 1979)
  1. Str, spreptomycin; Tri, trimethoprim; Tet, tetracycline; Kan, kanamycin.

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