DOI: 10.5061/dryad.xksn02vtc
title: Nitrogen fertilization nullifies host sanctions against non-fixing rhizobia and drives divestment from symbiosis in Lotus japonicus

description: Plants and animals house microbes that provide critical nutrients, but
 little is known about host control over microbial cooperation when
 resources are also accessed from the environment. Changes in nutrient
 access can challenge the host’s ability to detect and selectively reward
 beneficial partners, destabilizing symbiosis. Legumes acquire nitrogen
 from soil and from symbiosis with rhizobia, but it is unclear if extrinsic
 sources of nitrogen interfere with host control systems. We inoculated the
 legume, Lotus japonicus, with rhizobia bearing nitrogen fixation or
 nitrogen metabolism knockouts, and factorially varied molecular sources of
 nitrogen fertilizer. Lotus hosts selectively rewarded beneficial rhizobia
 and sanctioned non-fixing strains when extrinsic nitrogen was unavailable.
 Host benefits were undiminished when inoculated with rhizobia bearing
 nitrogen metabolism knockouts, suggesting redundancies in nitrogen
 provisioning systems. However, under nitrogen fertilization hosts did not
 discriminate between fixing and non-fixing rhizobia. Fertilized hosts
 formed miniaturized nodules housing limited rhizobia, divesting from
 symbiosis. Thus, sanctioning mechanisms rely on detection of nitrogen
 fixation differences among rhizobia strains and can break down in nitrogen
 rich environments. Nonetheless, divestment from symbiosis offers legumes
 robust host control, minimizing investment into rhizobia strains,
 irrespective of their capacity to provide benefit, when symbiosis services
 are not needed.
description: <strong>Sole nitrogen source fertilization
 experiment </strong> The <em>L.
 japonicus</em> ecotype Miyakojima MG-20 was used, inbred at the
 University of California Riverside to generate seeds following published
 protocols. The <em>M. loti</em> genotype MAFF303099 (hereafter
 MAFF) is a highly effective nitrogen fixing strain on <em>L.
 japonicus</em>. MAFF was modified to express dsRed, a red
 fluorescent protein visible under natural light. The near-isogenic strain
 STM6 (strain ID 17T02d02) was employed as a non-nitrogen fixing mutant.
 STM6 was generated by a signature tagged transposon inserted in the nifD
 gene, and was subsequently modified to express GFP. Both modified strains
 efficiently form nodules on <em>L. japonicus</em>, with MAFF
 providing substantial benefits to host growth while STM6 provides none.
 MAFF and STM6 were grown on agar plates with a modified arabinose
 gluconate medium (MAG). In mixed cultures, MAFF and STM6 can be easily
 differentiated by colony color. Other than differences in nitrogen
 fixation and colony color, previous work uncovered no differences between
 the strains in terms of their invitro growth rate or capacity to induce
 nodule formation under clonal conditions. <em>L.
 japonicus</em> plants thrive under organic and inorganic sources of
 extrinsic nitrogen (i.e., alanine, aspartic acid, ammonium sulfate,
 potassium nitrate) and plants fertilized with these nitrogen sources
 reduce investment in nodules infected with MAFF. In this
 experiment, <em>L. japonicus </em>was exposed to fertilization
 treatments and rhizobia that vary in nitrogen fixation to quantify effects
 on rhizobia fitness and examine how sanctions traits respond under
 fertilization<em>. L. japonicus</em> seedlings were exposed to
 clonal treatments of nitrogen fixing MAFF and non-fixing STM6, and mixed
 infections of both strains. Rhizobia cell proliferation, nodule biomass,
 and nitrogen fixation were quantified to examine rhizobia fitness, and
 fitness effects in planta under different treatments.
 <em>L. japonicus</em> seeds were surface sterilized
 in bleach (5% sodium hypochlorite), rinsed in sterile water, nick
 scarified, and planted under axenic conditions in bleach sterilized
 Ray-Leach SC10 pots (Stuewe & Sons, Corvallis, OR, USA) using an
 equal mix of coarse sand on top of a layer of fine calcined clay that was
 autoclave-sterilized (Pro League, Quickdry; Turface Athletics, Buffalo
 Grove, Illinois, USA). In a factorial experiment, plants were inoculated
 (i.e., on 12/21/2021) with one of four treatments including MAFF, STM6, an
 equal mix of both strains, and sterile ddH<sub>2</sub>O as a
 control. Inoculations were 5 ml and included 5 x
 10<sup>8</sup> cells, except for the water controls. High cell
 concentrations are needed to assure consistent nodulation in sterile
 coarse soils, likely because of low bacterial survival during inoculation.
 For mixed inoculation treatments 2.5 x 10<sup>8</sup> of each
 strain were combined, and the mixed inoculum was plated to empirically
 confirm the ratio of MAFF to STM6. All plants were supplemented weekly
 with 5 ml of Broughton & Dilworth solution, including key
 nutrients but lacking nitrogen. Hosts were fertilized with one of four
 sources of nitrogen dissolved in the Broughton & Dilworth
 solution, including alanine
 (C<sub>3</sub>H<sub>7</sub>NO<sub>2</sub>, 2.64 g/L), ammonium sulfate ((NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 3.92 g/L), aspartic acid (C<sub>4</sub>H<sub>7</sub>NO<sub>4</sub>, 3.95 g/L), or potassium nitrate (KNO<sub>3</sub>, 3 g/L). Concentrations were set to equalize the nitrogen moiety at levels previously shown to optimize host growth in the absence of rhizobia inoculation, thus attempting to isolate the growth effect of nitrogen. Fertilization occurred weekly, starting one day before inoculation (i.e., 12/20/2021) with 2 mL of fertilizer per plant and increasing by 1 mL per week until a maximum of 5 mL was reached 3 weeks later. This final level of fertilizer was maintained until the plants were harvested. Fourteen experimental blocks each contained a single plant replicate per each of the twenty treatments, including four inoculation treatments (i.e., MAFF, STM6, MAFF+STM6, water) crossed with five fertilization treatments (i.e., alanine, ammonium, aspartate, nitrate, no added nitrogen), totaling 280 plants. To minimize within-block variance, seedlings were assigned to blocks based on size at time of inoculation. Treatments within blocks were assigned randomly. Two blocks were harvested at three weeks post inoculation, the time when nodules first become visible on <em>L. japonicus </em>roots. Two additional blocks were harvested five weeks post inoculation, when most nodules have typically established. Data from early harvests were used to quantify rhizobia nodulation and growth effects over time in relation to fertilization treatments. The remaining ten blocks were harvested at week seven post inoculation to quantify root and shoot biomass, nodule count and nodule biomass, to quantify rhizobia fitness in planta (i.e., rhizobia population size in nodules), and to test for evidence of host sanctions (i.e., overrepresentation of the nitrogen fixing strain MAFF in the nodules of coinoculated plants). The mixed inoculum was plated to estimate the initial ratio of MAFF:STM6 by distinguishing colony color. During harvests, plants were removed from pots, washed free of soil, and dissected into root, shoot, and nodule portions. Nodules were counted and photographed. Within each inoculated treatment, four plants were randomly selected for quantitative culturing, wherein two nodules each were randomly sampled, surface sterilized in bleach for 30 seconds followed by 3 rinses in autoclave-sterilized ddH<sub>2</sub>O. Nodules were then crushed in 200µl of sterile ddH<sub>2</sub>O and the slurry was serially diluted to 0.5 x 10<sup>-6</sup> and 0.5 x 10<sup>-8</sup> on MAG-agar plates. Colony counts were averaged across at least two plate replicates per nodule to estimate rhizobia population size within each nodule. The ratio of MAFF:STM6 colonies was estimated from nodules plated from coinoculated plants and compared to the MAFF:STM6 ratio in the initial inoculum. Roots, shoots, and remaining nodules were weighed for biomass after being oven dried (&gt; 3 days, 60°C). Ten plants were removed from the analysis, 5 due to mortality, 5 due to contamination. Leaf ∂<sup>15</sup>N ‘atom percent difference’ was estimated as the percentage of <sup>15</sup>N atoms over total nitrogen in each sample and the values used as a measure of nitrogen fixation. Only plants harvested at seven weeks post inoculation were used. Individual leaves from each plant were oven dried, weighed, and placed in tin capsules for isotopic analysis at the UC Davis Stable Isotope Facility. <strong>Nitrogen metabolism knockout experiment </strong> Rhizobia strains with individual gene knockouts were inoculated onto <em>L. japonicus</em> MG-20 plants to test for changes in nodulation or host benefits. <em>M</em>. <em>loti </em>strains were selected from a large collection of single gene mutants, focusing on knockouts of genes whose loss is predicted to disrupt the symbiotic transfer of nitrogen from rhizobia to the host. No nitrogen fertilizer was used so we could examine effects of these mutations when hosts have no other nitrogen source. Five <em>M. loti</em> strains were tested, including MAFF as a control, and four near isogenic, signature-tagged mutants. <em>M. loti </em>03T03g02 has a knockout for a periplasmic amino acid ABC transporter (mll3861), a protein similar to one in <em>Rhizobium</em> that transfers alanine or aspartate to the host. <em>M. loti </em>27T02f13, has a knockout for L-2,4-diaminobutyric acid transaminase (mlr5943), an enzyme involved in alanine synthesis that is upregulated in nodules compared to growth on media, suggesting importance for nitrogen transfer to the host during symbiosis. The last two mutants carry knockouts for paralogs of aspartate aminotransferase, which produces aspartate from oxaloacetate, predicted to be a mechanism to generate nitrogen usable to the host. <em>M. loti </em>8T11d03 carries a knockout for mlr5883, encoded on the symbiosis island, and whose expression is upregulated <em>in planta</em> during symbiosis, whereas <em>M. loti </em>20T04d01 has a knockout for the other copy, mlr2541, which is encoded on the bacterial chromosome, and is constitutively expressed. None of the mutants have been previously tested on <em>L. japonicus.</em> Fifteen replicate plants per treatment were singly inoculated with one of the mutant<em> </em>strains, with MAFF, or with sterile water, to examine nodulation traits and growth effects, totaling 90 plants. Plants were prepared, inoculated, and maintained as outlined in the fertilization experiment, with the modification of only receiving the nitrogen-free Broughton & Dilworth (micronutrient solution). Plants were harvested at seven weeks after inoculation to quantify root and shoot biomass, nodule count, and nodule biomass. <strong>Symbiosis trait measurements</strong> Host biomass was used to estimate host growth in different treatments. Nodulation was used to estimate host investment into symbiosis, quantified as the number of nodules formed, their mean biomass, or total nodule biomass per plant. Host investment into symbiosis was also calculated by dividing the dry nodule biomass of each plant over the total plant biomass. Rhizobia population size in nodules, a proxy of rhizobia fitness in planta, was calculated by quantitative culturing of nodules of individual plants. The ratio of MAFF:STM6 colonies cultured from coinoculated plants was used to analyze relative fitness of the beneficial strain and to test for sanctions. Nitrogen fixation was measured using the abundance of <sup>15</sup>N in the plant shoots from different treatments. <strong>Data analysis</strong> Statistical analyses were conducted using linear mixed models in JMP (Ver. 16.0.0). All data distributions were tested for normality using the Shapiro-Wilk test, as this is a prerequisite for parametric statistics. Data were transformed when necessary to achieve normal distributions to satisfy this requirement. In some cases, log or square root transformations were used. Full models, transformations, and random versus fixed factors are all listed (Data Supplement). Tukey’s HSD tests were used to test for significant differences among groups in a post hoc manner, after observing a significant effect. The ∂<sup>15</sup>N of each sample was calculated by comparing <sup>15</sup>N abundance expressed as parts per thousand to atmospheric N<sub>2</sub>. The values were then used to compare values of ∂<sup>15</sup>N between treatments by subtracting the mean sample atom % <sup>15</sup>N of uninoculated plants from the sample atom % <sup>15</sup>N of inoculated plants. When plants incorporate fixed nitrogen, leaf tissues exhibit a decrease in <sup>15</sup>N (compared to uninfected plants) due to isotopic fractionation by rhizobia. To test for sanctions, a chi-square test was used to test whether MAFF was overrepresented in nodules of coinoculated plants, relative to the proportion of MAFF in the inoculum.
description: # Nitrogen fertilization nullifies host sanctions against non-fixing
 rhizobia and drives divestment from symbiosis in Lotus japonicus Dataset
 DOI:
 [10.5061/dryad.xksn02vtc](https://datadryad.org/submission/10.5061/dryad.xksn02vtc) ## Description of the data and file structure **Nitrogen fertilization nullifies host sanctions against non-fixing rhizobia and drives divestment from symbiosis in *Lotus japonicus*** ### Host Biomass data * Plant_ID: Unique identifier for the focal plant (includes block number followed by the plant replicate number) * Block: Block in which the focal plant was grown within the greenhouse * Strain_Treatment: Category of strain (or strains) to which the focal plant was assigned; Fix+ = the nitrogen fixing strain MAFF303099; Fix- = the near isogenic non-fixing mutant STM6l; Coinoculant = co-inoculation with strains both strains; No Inocula = inoculation with sterile water * Modified_Strain_Treatment: Same as above, but changes categories of plants that did not form nodules into the ‘No inocula’ category * Nutrient_Color: Color on labeling tape on plant in the greenhouse to identify the fertilizer treatment used * Nutrient_source: The form of nitrogen used to fertilize the plants including alanine (C3H7NO2, 2.64 g/L), ammonium sulfate ((NH4)2SO4, 3.92 g/L), aspartic acid (C4H7NO4, 3.95 g/L), or potassium nitrate (KNO3, 3 g/L). * Organic: Describes whether the form of nitrogen used is organic, including alanine (C3H7NO2, 2.64 g/L) and aspartic acid (C4H7NO4, 3.95 g/L), or not, including potassium nitrate (KNO3, 3 g/L) and ammonium sulfate ((NH4)2SO4, 3.92 g/L). * Dead: Categorizes whether the plant died in the course of the experiment (YES) or if it survived to the harvest (NO). * Num_Nodules: The count of nodules dissected from each plant * Num_Nods_MinCulture: The count of nodules quantitatively cultured from each plant * Harvest_date: Date the plant was dissected into root, shoot, and nodule portions * Shoot_Weight(mg): Shoot dry mass (mg) of focal plant * Square Root[Shoot_Weight (mg)]: Square root transformation of Shoot dry mass (mg) of focal plant * Root_Weight (mg):  Root dry mass (mg) of focal plant * Square Root[Root_Weight (mg)]: Square root transformation of Root dry mass (mg) of focal plant * Nodule_Weight (mg): Total nodule dry mass (mg) of focal plant * Nod_Weight_Adjusted (mg): Adjustment to total nodule dry mass (mg) of focal plant from inferred mass of nodules removed for culturing. * Square Root[Nod_Weight_Adjusted (mg)]:  Square root transformation of adjusted nodule biomass. * Avg_Nod_Weight (mg): Total nodule dry mass (mg) of focal plant divided by the number of nodules from that plant (average nodule biomass) * Log[Avg_Nod_Weight (mg)]: Log transformation of average nodule biomass * Total_Weight (mg): Sum of shoot, root, and nodule biomass (mg) of a focal plant (total weight) * Square Root[Total_Weight (mg)]: Square root transformation of total weight * Root/Shoot Ratio: Root biomass divided by shoot biomass of a focal plant * Log[Root/Shoot Ratio]: Log transformation of Root/Shoot Ratio * Host_Relative_Growth: Unitless measure of relative host growth. Relative growth response of hosts to infection was calculated as the proportional difference in total dry plant biomass between inoculated hosts (infected) relative to the (uninoculated) control plants. * Log[Host_Relative_Growth]: Log transformation of relative growth response * Nodule/Total Biomass: Total nodule dry mass (mg) of focal plant divided by its total biomass * Log(Nodule/Total Biomass): Log transformation of Nodule/Total Biomass * Cultured: Indicates whether nodules were quantitatively cultured from the focal plant \**************************************** ### Nodule Culture data * Plant_ID:  Unique identifier for the focal plant (includes block number followed by the plant replicate number) * Block: Block in which the focal plant was grown within the greenhouse * Inoc: Category of strain (or strains) to which the focal plant was assigned; Fix+ = the nitrogen fixing strain MAFF303099; Fix- = the near isogenic non-fixing mutant STM6; Coinoculant = co-inoculation with strains both strains; No Inocula = inoculation with sterile water * N_Source: The form of nitrogen used to fertilize the plants including alanine (C3H7NO2, 2.64 g/L), ammonium sulfate ((NH4)2SO4, 3.92 g/L), aspartic acid (C4H7NO4, 3.95 g/L), or potassium nitrate (KNO3, 3 g/L). * Nitrogen_vs_No_Nitrogen: Fertilizer treatments are categorized as to whether they include nitrogen (alanine, ammonia, aspartic acid, nitrate) or do not (i.e., water control) * Nodule: Individual cultured nodules within a plant are assigned letter identifies, i.e., A, B, etc. * MAFF (Number Rhizobia): Estimated colony forming units (CFUs) of the nitrogen fixing strain MAFF303099 in each focal cultured nodule * Square Root[MAFF (Number Rhizobia)]: Square root transformation of MAFF CFUs (see above) * STM (Number Rhizobia): Estimated colony forming units (CFUs) of the non-fixing mutant strain STM6 in each focal cultured nodule * Square Root[STM (Number Rhizobia)]: Square root transformation of STM CFUs (see above) * TOTAL (Number Rhizobia): Estimated colony forming units (CFUs) in each focal cultured nodule * Square Root[TOTAL (Number Rhizobia)]: Square root transformation of TOTAL CFUs (see above) \**************************************** ### N15 data * Plant_ID: Unique identifier for the focal plant (includes block number followed by the plant replicate number) * Block: Block in which the focal plant was grown within the greenhouse * N-Treatment: The form of nitrogen used to fertilize the plants including alanine (C3H7NO2, 2.64 g/L), ammonium sulfate ((NH4)2SO4, 3.92 g/L), aspartic acid (C4H7NO4, 3.95 g/L), or potassium nitrate (KNO3, 3 g/L). * Inocula: Category of strain (or strains) to which the focal plant was assigned; Fix+ = the nitrogen fixing strain MAFF303099; Fix- = the near isogenic non-fixing mutant STM6l; Coinoculant = co-inoculation with strains both strains; No Inocula = inoculation with sterile water * Mod Inocula: Same as above, but changes categories of plants that did not form nodules into the ‘None’ category (i.e., No inocula) * MAFF: Categorization of strain treatments, whether the nitrogen fixing strain MAFF is present (i.e., MAFF) or absent (i.e., None) * δ15NAir (‰): Leaf ∂15N ‘atom percent difference’ was estimated for each focal plant as the percentage of 15N atoms over total nitrogen in each sample and the values used as a measure of nitrogen fixation \**************************************** ### Mutant data * Plant ID: Unique identifier for the focal plant (includes block number followed by the plant replicate number) * Block: Block in which the focal plant was grown within the greenhouse * Mutant Inocula: Five *M. loti* strains were tested, including the nitrogen fixing strain MAFF303099 as a control, and four near isogenic, signature-tagged mutants. *M. loti* 03T03g02 has a knockout for a periplasmic amino acid ABC transporter (mll3861), a protein similar to one in *Rhizobium* that transfers alanine or aspartate to the host. *M. loti* 27T02f13, has a knockout for L-2,4-diaminobutyric acid transaminase (mlr5943), an enzyme involved in alanine synthesis that is upregulated in nodules compared to growth on media, suggesting importance for nitrogen transfer to the host during symbiosis [56]. The last two mutants carry knockouts for paralogs of aspartate aminotransferase, which produces aspartate from oxaloacetate, predicted to be a mechanism to generate nitrogen usable to the host. *M. loti* 8T11d03 carries a knockout for mlr5883, encoded on the symbiosis island, and whose expression is upregulated *in planta* during symbiosis, whereas *M. loti* 20T04d01 has a knockout for the other copy, mlr2541, which is encoded on the bacterial chromosome, and is constitutively expressed. None of the mutants have been previously tested on *L. japonicus.* * Nodule Number: The count of nodules dissected from each plant * Shoot Weight (mg): Shoot dry mass (mg) of focal plant * Root Weight (mg): Root dry mass (mg) of focal plant * Nodule Weight (mg): Total nodule dry mass (mg) of focal plant * Total Weight (mg): Total plant dry mass (mg) of focal plant * Mean Nodule Weight (mg): Total nodule dry mass (mg) of focal plant divided by the number of nodules from that plant (average nodule biomass). NA means that no data were collected. * Nodule/Total Weight: Total nodule dry mass (mg) of focal plant divided by its total biomass. NA means that no data were collected. * Log[Nodule/Total Weight]: Log transformation of Total nodule dry mass. NA means that no data were collected. * Host Relative Growth: Unitless measure of relative host growth. Relative growth response of hosts to infection was calculated as the proportional difference in total dry plant biomass between inoculated hosts (infected) relative to the (uninoculated) control plants. NA means that no data were collected. * Notes: Indicates additional information relating to the condition of plants. NA means that no notes exist for this row. \**************************************** ### Early Harvest 2 data * Block: Block in which the focal plant was grown within the greenhouse * Plant: Replicate number of focal plant * Inoc: Category of strain (or strains) to which the focal plant was assigned; Fix+ = the nitrogen fixing strain MAFF303099;  Fix- = the near isogenic non-fixing mutant STM6l; Coinoculant = co-inoculation with strains both strains; No Inocula = inoculation with sterile water * N-Source: The form of nitrogen used to fertilize the plants including alanine (C3H7NO2, 2.64 g/L), ammonium sulfate ((NH4)2SO4, 3.92 g/L), aspartic acid (C4H7NO4, 3.95 g/L), or potassium nitrate (KNO3, 3 g/L). * Shoots (g): Shoot dry mass (g) of focal plant * Roots (g): Root dry mass (g) of focal plant * Total (g): Total dry mass (g) of focal plant (root + shoot)