Rapid evolution of bacterial mutualism in the plant rhizosphere

Bacterial strain and growth conditions

We used Pseudomonas protegens (formerly Pseudomonas fluorescens)⁵⁵ CHA0 as a model strain, which was initially isolated from tobacco roots⁵⁶. The strain was chromosomally tagged with GFP and a kanamycin resistance cassette to enable specific tracking of the strain and detection of contaminations¹⁹. This bacterium has the genetic potential to produce various bioactive metabolites, including the plant hormone indole-3-acetic acid (IAA), antimicrobial compounds and lytic enzymes²⁵. Prior to the experiment, bacteria were grown for 48 h on a King′s medium B⁵⁷ (KB) agar plate supplemented with 50 µg ml⁻¹ kanamycin, a single colony was randomly picked and grown for 12 h in KB at 28 °C with agitation. The cell culture was then washed for three times in 10 mM MgSO₄ and adjusted to 10⁷ cells ml⁻¹ and used as inoculant for all plants. This inoculant was also stored at −80 °C as frozen ancestral stock, from which ʻAncestorʼ isolates were picked in later experiments.

Host plant and growth conditions

We used Arabidopsis thaliana ecotype Col-0 as a model host plant. Surface-sterilized seeds were first sown in Petri dishes with agar-solidified (1.5% agar (w/v)) modified Hoagland′s medium: (KNO₃ (3 mM), MgSO₄ (0.5 mM), CaCl₂ (1.5 mM), K₂SO₄ (1.5 mM), NaH₂PO₄ (1.5 mM), H₃BO₃ (25 μM), MnSO₄ (1 μM), ZnSO₄ (0.5 μM), (NH₄)₆Mo₇O₂₄ (0.05 μM), CuSO₄ (0.3 μM), MES (2.5 mM) and 50 μM Fe(III)EDTA, pH = 5.8) and stratified for 2 days at 4 °C after Petri dishes were positioned vertically and transferred to a growth chamber (20 °C, 10 h light/14 h dark, light intensity 100 μmol m⁻² s⁻¹). After 2 weeks of incubation, two seedlings were transferred to closed and sterile ECO2 boxes (http://www.eco2box.com/ov80xxl_nl.htm) for selection experiment. The ECO2 boxes were filled with 260 g of dry, carbon-free silver sand that was previously washed with MilliQ water to remove dissolvable chemical elements and heated to 550 °C for 24 h to remove remaining organic material. Prior to transplantation the sand was amended with 13 ml of modified Hoagland medium.

Design of the selection experiment

The selection experiment was conducted in a gnotobiotic system to remove confounding effects that may emerge as a result of competitive interactions with other microorganisms, and to place the focus on plant-mediated selective pressures. Moreover, we allowed only the bacteria to evolve during the experiment and used new clonal plants at every bacterial transfer. We set up five independent plant–bacterium replicate lines, which were grown for six independent plant growth cycles (see Fig. S1 for an overview of the experimental design). The experiment was started by inoculating 10⁶ cells of the stock P. protegens CHA0 culture (from here on abbreviated as ʻancestorʼ) into the rhizosphere of 2-week-old A. thaliana seedlings growing in sterile silver sand within ECO2 boxes (two plants per replicate selection line). Inoculated plants were then grown for 4 weeks (20 °C, 10 h light/14 h dark, light intensity 100 μmol m⁻² s⁻¹) after which the plant growth cycle was terminated and root-associated bacteria were harvested by placing the roots of both plants into a 1.5 ml Eppendorf tubes filled with 1 ml 10 mM MgSO₄ and two glass beads. Rhizosphere bacteria were suspended into the liquid using a TissueLyser II at a frequency of 20 s⁻¹ for 1 min after which bacterial cell densities were determined using flow cytometry (BD Accuri™ C6 Plus, thresholds for FSC: 2000, SSC: 8000). After this, 10⁶ cells were inoculated to the rhizosphere of new A. thaliana plants to initiate the next plant growth cycle. Possible contaminations were checked by plating the suspension on 3 g l⁻¹ tryptic soy agar (TSA) plates and it was verified that all colonies carried the GFP marker gene, as observed under UV light.

Bacterial life-history traits measurements

Individual bacterial colonies were isolated from all replicate plant selection lines for life-history measurements at the end of the second, fourth and sixth plant growth cycle by dilution plating the rhizosphere suspension on 3 g l⁻¹ TSA plates. After incubation at 28 °C for 24 h, 16 colonies were randomly picked from each replicate selection lines resulting in a total of 240 evolved and 16 ancestral colonies. All these colonies were characterized for a set of key bacterial life-history traits representative of growth, stress resistance and traits linked with plant–microbe interactions.

1. a.

   Bacterial growth yield in KB medium

   All the bacterial isolates were grown in 96-well plates with 160 µl 1/3 strength liquid KB, at 20 °C without shaking. Bacterial yield was determined as the maximum optical density at 600 nm after 3 days of growth using a spectrophotometer (SPECTROstar Nano).

2. b.

   Bacterial stress resistance

   We measured bacterial resistance to a range of different stresses using various 96-well microplate assays. Abiotic stress resistance was determined by growing bacteria in 160 µl of 1 g l⁻¹ TSB containing 0.0025% H₂O₂ (oxidative stress), 15% polyethylene glycol (PEG)−6000 (water potential stress) or 2% NaCl (salt stress). We used resistance to antibiotics commonly produced by rhizosphere microorganisms as indicator of biotic stress resistance. Antibiotic resistance was tested in 160 µl of 1 g l⁻¹ TSB supplemented with 1 µg ml⁻¹ streptomycin, 1 µg ml⁻¹ tetracycline, or 5 µg ml⁻¹ penicillin, respectively. Bacterial growth were determined after 3 days of growth at 20 °C without shaking as optical density at 600 nm.

3. c.

   Traits linked with plant–microbe interactions

   P. protegens CHA0 harbours several traits that are linked to plant growth including production of antibiotics and plant hormones. To assess these traits, we grew each bacterial colony in 96-well plates containing 160 µl of 1/3 strength liquid KB per well at 20 °C without shaking for 72 h. Cell-free supernatants were obtained by filter sterilization (0.22 µm) using Multiscreen HTS 96-well filtration plates (1000 × g, 30 min), which were used to measure the production of the plant hormone auxin (Indole-3-acetic acid (IAA)), iron-chelating siderophores and proteolytic activity. Furthermore, we also measured antifungal and antibacterial activity of all colonies.

IAA detection

The production of the plant hormone auxin was determined with a colorimetric test⁵⁸. Briefly, 30 µl P. protegens CHA0 cell-free filtrate was incubated with 30 µl R1 reagent (12 g l⁻¹ FeCl₃, 7.9 M H₂SO₄) for 12 h in the dark and optical density read at 530 nm of the colorimetric complex was used as a measurement of IAA concentration.

Siderophore activity

Iron-chelating ability was measured as a proxy for siderophore production⁵⁹. To this end, 100 µl of P. protegens CHA0 cell-free filtrate was mixed with 100 µl of modified CAS solution (with 0.15 mM FeCl₃) and optical density read at 630 nm after 3 h of incubation was used as a proxy of siderophore production. The iron-chelating ability was calculated based on the standard curve based on modified CAS assay solution with a range of iron concentration (0, 0.0015, 0.003, 0.006, 0.009, 0.012, 0.015 mM FeCl₃).

Proteolytic activity

The proteolytic activity assay we used was adapted from Smeltzer et al.⁶⁰. Briefly, 15 µl of P. protegens CHA0 cell-free filtrate was incubated with 25 µl of azocasein (2% w/v in 50 mM Tris-HCl pH 8.0) at 40 °C for 24 h. One hundred and twenty-five of 10% w/v cold trichloroacetic acid (TCA) was added to precipitate superfluous azocasein, and then 100 µl supernatant was neutralized with 100 µl of 1 M NaOH after centrifugation at 5000g for 30 min. Optical density read at 440 nm was used as a proxy of exoprotease activity.

Tryptophan side chain oxidase (TSO) activity

TSO activity, an indicator of quorum sensing activity in P. protegens CHA0, was measured based on an modified established colorimetric assay⁶¹: Three-day-old bacterial cultures grown in 1/3 strength liquid KB were mixed at a 1:1 ratio with a reagent solution (5 g l⁻¹ SDS, 37.6 g l⁻¹ glycine 2.04 l⁻¹ g tryptophan, pH 3.0) and TSO activity was measured as optical density at 600 nm after overnight incubation.

Biofilm formation

We quantified bacterial biofilm formation using a standard protocol⁶². Briefly, bacteria were grown at 20 °C for 72 h in 160 µl 1 g l⁻¹ TSB in a 96-well microtiter plate with TSP lid (TSP, NUNC, Roskilde, Denmark). Planktonic cells were removed by immersing the lid with pegs three times in phosphate-buffered saline solution (PBS). Subsequently, the biofilm on the pegs was stained for 20 min in 160 µl 1% crystal violet solution. Pegs were washed five times in PBS after which the crystal violet was extracted for 20 min from the biofilm in a new 96-well microtiter plate containing 200 µl 96% ethanol per well. Biofilm formation was defined as the optical density at 590 nm of the ethanol extracted crystal violet⁶³.

Inhibition of other microorganisms

Antimicrobial activity was defined as the relative growth of the target organism in P. protegens supernatant compared to the control treatment. Antifungal activity of the cell-free supernatant was assessed against the ascomycete Verticillium dahliae. The fungus was grown on potato dextrose agar at 28 °C for 4 days, after which plugs of fungal mycelium were incubated in potato dextrose broth medium at 28 °C and gentle shaking for 5 days. Fungal spores were collected by filtering out the mycelium from this culture over glass wool. Subsequently, spores were washed and resuspended in water and the OD₅₉₅ of the suspension was adjusted to 1. Five microlitres of this spore suspension was then inoculated with 15 µl P. protegens CHA0 cell-free filtrate and incubated in 160 µl of 1 g l⁻¹ PDB medium for 2 days at 20 °C in 96-well plates. Fungal growth was measured as optical density at 595 nm after 2 days of growth and contrasted with the growth in the control treatment (PDB medium without P. protegens supernatant). Antibacterial activity was determined using the plant pathogen Ralstonia solanacearum as a target organism. R. solanacearum was grown in 160 µl of 1 g l⁻¹ TSB medium supplemented with 15 μl of P. protegens CHA0 cell-free filtrate or 15 µl of 1/3 strength liquid KB as a control for 2 days at 20 °C. R. solanacearum growth was measured as optical density at 600 nm.

Determining changes in P. protegens CHA0 interactions with A. thaliana after the selection experiment

Based on the life-history trait measurements, five distinct bacterial phenotypes were identified using K-means clustering analysis (Fig. S2). In order to assess whether phenotypic changes reflected shifts in the strength and type of plant–bacterium interaction, we chose five isolates from each bacterial phenotype group representing each replicate selection line and five ancestral isolates for further measurements (a total of 30 isolates, Table S2).

Effects of ancestor and evolved bacteria on plant performance

For each isolate we measured root colonizing ability and impact on plant performance. All 30 bacterial isolates were incubated overnight in 1/3 KB strength liquid at 20 °C. The culture was centrifuged twice for 5 min at 5000 × g and the pellet was washed and finally resuspended in 10 mM MgSO₄. The resulting suspension was adjusted to an OD₆₀₀ of 0.01 for each strain⁶⁴. Ten microlitres of the bacterial suspension (or 10 mM MgSO₄ as a control) was applied to the roots of three 10-day old sterile Arabidopsis thaliana Col-0 seedlings (excluding 2 days of stratification at 4 °C) grown on vertically positioned Petri dishes with agar-solidified (1.5% agar (w/v)) modified Hoagland′s medium (n = 3 biological plant replicates, each containing 3 seedlings). Plants were grown for 14 days before harvesting. Plants were photographed before and 14 days after bacterial inoculation.

Bacterial effects on plant health were quantified as leaf ʻgreennessʼ as the presence of ancestral strain was observed to lead to bleaching and loss of chlorophyll in A. thaliana leaves. The ʻgreennessʼ was quantified from photographs by measuring the number of green pixels. To this end, photographs were first transformed in batch using Adobe Photoshop 2021 by sequentially selecting only green areas followed by thresholding balancing green tissue over background noise (Level 80). This resulted in black-and-white images for further analysis, and the mean number of white pixels per fixed-sized region-of-interest of the aboveground tissue was subsequently determined as ʻgreennessʼ using ImageJ (version 1.50i). The numbers of lateral roots and the primary root length were also measured using ImageJ. The root morphology data measured at the end of the experiment was normalized with the data collected at the time of inoculation for each individual seedling.

To determine shoot biomass, the rosette of each plant was separated from the root system with a razor blade and weighted. The roots were placed into a pre-weighted 1.5 ml Eppendorf tubes to quantify the root biomass. To determine the bacterial abundance per plant, these tubes were subsequently filled with 1 ml 10 mM MgSO4 buffer solution and two glass beads. The rhizosphere bacteria were suspended into the buffer solution using a TissueLyser II at a frequency of 20 s⁻¹ for 1 min after which bacterial densities were determined using flow cytometry (BD Accuri™ C6 Plus, thresholds for FSC: 2000, SSC: 8000). Shoot biomass, root biomass, root length and number of lateral roots were used in a principal component analysis (PCA) to calculate an overall impact of the bacteria on plant performance (Fig. 2e). The first principal component (PC1) explained 79.9% of the variation and was normalized against the control treatment to be used as a proxy of ʻPlant performanceʼ in which positive values reflect plant growth promotion and negative values plant growth inhibition.

Root derived carbon source utilization

To measure changes in bacterial growth on potential root derived carbon sources, we measured the growth of all 256 isolates using modified Ornston and Stanier (OS) minimal medium⁶⁵ supplemented with single carbon sources at a final concentration of 0.5 g l⁻¹ in 96-well plates containing 160 µl carbon supplemented OS medium per well. The following carbon sources were selected based on their relatively high abundance in Arabidopsis root exudates²¹: alanine, arabinose, butyrolactam, fructose, galactose, glucose, glycerol, glycine, lactic acid, putrescine, serine, succinic acid, threonine and valine. Bacterial growth was determined by measuring optical density at 600 nm after 3 days incubation at 20 °C.

GUS histochemical staining assay and bacterial growth under scopoletin stress

To investigate effects of the ancestor and evolved strains of P. protegens CHA0 on expression of MYB72, we applied a GUS histochemical staining assay to the 30 selected isolates (Table S2). MYB72 is a transcription factor involved in production of the coumarin scopoletin in Arabidopsis roots and specific rhizobacteria can upregulate expression of MYB72 in the roots⁶⁶. Scopoletin is an iron-mobilizing phenolic compound with selective antimicrobial activity²². Seedlings of the A. thaliana MYB72_pro:GFP-GUS²⁴ reporter line were prepared as described above. Seven-day-old seedlings were inoculated directly below the hypocotyls with 10 μl of a bacterial suspension (OD₆₆₀ = 0.1)²⁴. At 2 days after inoculation, the roots were separated from the shoots and washed in MilliQ water (Milliport Corp., Bedford, MA) to remove all the adhered bacteria. GUS staining of the roots was performed in 12-well microtiter plates where each well contained roots of 5–6 seedlings and 1 ml of freshly prepared GUS substrate solution (50 mM sodium phosphate with a pH at 7, 10 mM EDTA, 0.5 mM K4[Fe(CN)6], 0.5 mM K3[Fe(CN)6], 0.5 mM X-Gluc, and 0.01% Silwet L-77)⁶⁷. Plates were incubated in the dark at room temperature for 16 h. The roots were fixed overnight in 1 ml ethanol:acetic acid (3:1 v/v) solution at 4 °C and transferred to 75% ethanol. Then the pictures of each microtiter plates were taken, and GUS activity was quantified by counting the number of blue pixels in each well of the microtiter plates using image analysis in ImageJ (version 1.52t). To assess the effects of scopoletin on ancestral and evolved P. protegens CHA0 isolates, we applied a sensitivity assay to the 30 selected isolates (Table S2). In brief, growth of bacterial isolates was measured in 1 g l⁻¹ TSB medium (160 µl) supplemented with scopoletin at final concentrations of 0 µM (control), 500 µM, 1000 µM, and 2 mM using optical density at 600 nm after 72 h incubation at 20 °C without shaking in 96-well microtiter plates. Maximal effect (Emax) of scopoletin was calculated via R package ʻGRmetricsʼ⁶⁸ as an indication of scopoletin tolerance.

Whole-genome sequencing

All 30 isolated phenotypes were whole genome sequenced to identify possible mutations and affected genes. To this end, isolates were cultured overnight at 28 °C in 1/3 strength liquid KB. Chromosomal DNA was isolated from each culture using the GenElute™ Bacterial Genomic DNA Kit Protocol (NA2100). DNA samples were sheared on a Covaris E-220 Focused-ultrasonicator and sheared DNA was then used to prepare Illumina sequencing libraries with the NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs. France) and the NEBNext® Multiplex Oligos for Illumina® (96 Index Primers). The final libraries were sequenced in multiplex on the NextSeq 500 platform (2 × 75 bp paired-end) by the Utrecht Sequencing Facility (http://www.useq.nl) yielding between 1.0 and 6.4 million reads per sample equivalent to ~10–70-fold coverage (based on comparison with the original 6.8 Mbp reference genome NCBI GenBank: CP003190.1).

Variant calling analysis

We first constructed an updated reference genome of P. protegens CHA0, carrying the GFP marker gene on its chromosome, from the ancestral strain using the A5 pipeline with default parameters⁶⁹. The input dataset for this sample consisted of 3,1M reads and totals an approximate 34-fold coverage. The size of the updated reference genome is 6.8 Mbp, with a G + C content of 63.4%, and it comprises 80 scaffolds, with a N₅₀ value of 343 kbp. We subsequently used PROKKA⁷⁰ (version 1.12; https://github.com/tseemann/prokka) for full annotation of the updated reference genome, and this resulted in the identification of 6147 genes. The updated genome is deposited in NCBI GenBank with following reference: RCSR00000000.1.

Having established the ancestral genome sequence, we subsequently used Snippy (version 3.2-dev; https://github.com/tseemann/snippy) to identify and functionally annotate single-nucleotide polymorphisms and small insertions and deletions (indels) for each individual strain. In addition, we investigated the breadth of coverage for each gene per sample with BedTools⁷¹ to identify genes with large insertions or deletions. An overview of the polymorphisms is shown in Supplementary Table S3. Raw sequencing data for this study are deposited at the NCBI database under BioProject PRJNA473919.

Relative competitive fitness of gac mutants measured in vivo and in vitro

The relative competitive fitness of selected gac mutants was measured in direct competition with their direct ancestors both in vivo in the rhizosphere of A. thaliana and in vitro in different standard culture media. Relative fitness was measured as deviation from initial 1:1 ratio of bacterial clone pairs based on PCR-based high-resolution melting profile (RQ-HRM) analysis. Three pairs of isolates were selected: (A) evolved gacA ID 242 (genotype oafA^Y335X ∙ RS17350^A77A.fsX14 ∙ gacA^D49Y) and its direct ancestral genotype 133 (genotype oafA^Y335X ∙ RS17350^A77A.fsX14) from evolutionary line 1; (B) evolved gacA ID 220 (genotype galE^V32M ∙ accC^E413K ∙ gacA^D54Y) and its direct ancestral genotype 28 (genotype galE^V32M ∙ accC^E413K) from line 2; (c) evolved gacS ID 222 (genotype oafA^K338S.fsX18 ∙ gacS^G27D) and its direct ancestral genotype 66 (genotype oafA^K338S.fsX18) from line 3. Bacterial isolates were first grown overnight in KB medium at 28 °C, centrifuged at 5000g for 10 min and the pellet resuspended in 10 mM MgSO₄. This washing procedure was repeated twice. The resulting bacterial suspensions were diluted to OD₆₀₀ = 0.05. The initial inoculum for the competition assays was then generated by mixing equal volumes of evolved and ancestral competitors in a ratio of 1:1.

Measuring competitive fitness in A. thaliana rhizosphere

This assay was performed on the roots of 10-day old A. thaliana seedlings grown on full strength Hoagland agar plates, which were prepared as described earlier. Twenty microlitres of the initial inoculum, containing a total of 10⁶ bacterial cells, was inoculated on to the root–shoot junction of each seedling. After 14 days of growth, bacterial populations were isolated from the roots and stored at −80 °C in 42.5% glycerol for relative abundance measurements.

Measuring competitive fitness in culture media

Competition assays were also performed in three commonly used nutrient-rich growth media: KB, LB and TSB. KB contained 20 g proteose peptone, 1.5 g MgSO₄.7H₂O, 1.2 g KH₂PO₄ and 10 g glycerol per litre and the pH was adjusted to 7.3 ± 0.2. TSB contained 30 g tryptic soy broth per litre and pH was adjusted to 7.3 ± 0.2. LB contained 10 g peptone, 5 g yeast extract and 5 g NaCl per litre. Twenty microlitres inoculum of competing strains, containing about 10⁶ bacterial cells, were added into wells containing 140 μl fresh medium in a 96-well plate. The microplates were incubated at 28 °C without shaking for 48 after 80 μl sample was harvested and stored at −80 °C in 42.5% glycerol from each well for relative abundance measurements.

RQ-HRM assay for quantifying changes in genotype frequencies after competition

We used a high-resolution melting (HRM) curve profile assay with integrated LunaProbes to quantify the ratio of mutant to wild-type genotypes^72,73,74. The probes and primers used in this study are listed in Table S4. Primers were designed using Primer3. Probes were designed with the single-nucleotide polymorphism (SNP) located in the middle of the sequence, and the 3′ end was blocked by carbon spacer C3. The primer asymmetry was set to 2:1 (excess primer: limiting primer) in all cases. Pre-PCR was performed in a 10-μl reaction system, with 0.25 μM excess primer, 0.125 μM limiting primer, 0.25 μM probe, 0.5 μl bacterial sample culture (100-fold diluted saved sample, OD₆₀₀ is about 0.01), 1× LightScanner Master Mix (BioFire Defense). DMSO with the final concentration 5% was supplemented in all reactions to ensure the targeted melting domains are within the detection limit of the LightScanner (Idaho Technology Inc.). Finally, MQ water was used to supplement up to 10 μl. A 96-well black microtiter plate with white wells was used to minimize background fluorescence. Before amplification, 25 μl mineral oil was loaded in each well to prevent evaporation, and the plate was covered with a foil seal to prevent the degradation of fluorescent molecules. Amplification was initiated by a holding at 95 °C for 3 min, followed by 55 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s and then kept at 72 °C for 10 min. After amplification, samples were heated in a ThermalCycler (Bio-Rad) shortly to 95 °C for 30 s to denature all double-stranded structures followed by a rapid cooling to 25 °C for 30 s to facilitate successful hybridization between probes and the target strands. The plate was then transferred to a LightScanner (Idaho Technology Inc.). Melting profiles of each well were collected by monitoring the continuous loss of fluorescence with a steady increase of the temperature from 35 to 97 °C with a ramp rate of 0.1 °C/s. The relative quantification was based on the negative first derivative plots using software MATLAB. The areas of probe-target duplexes melting peaks were auto-calculated by ʻAutoFit Peaks I Residualsʼ function in software PeakFit (SeaSolve Software Inc.). The mutant frequency X was calculated using the formula shown below:

To validate the RQ-HRM method, standard curves were generated by measuring mixed samples with known proportions of mutant templates: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100%. Measurements for each sample were done in triplicate. Linear regression formula of each mutant between actual frequencies and measured frequencies are shown in Fig. S7. The high R² values, and nearly equal to 1 slope values of these equations, confirmed that the RQ-HRM method can accurately detect mutantsʼ frequency in a mixed population.

The relative fitness of the evolved strains was calculated according to previous studies using the following equation^75,76:

where X₀ is the initial mutant frequency; (1−X₀) the initial ancestor frequency; X₁ the final mutant frequency; and (1−X₁) is the final ancestor frequency.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Source: Ecology - nature.com