Materials and Reagents

2-mL microcentrifuge tubes (Sarstedt, catalog number: 72.695.400)
5-mL tubes (Sarstedt, catalog number: 72.701)
Petri dishes 92 × 16 mm (Sarstedt, catalog number: 82.1473)
1-mL Blunt syringe (SOFT-JECT HSW, catalog number: 5010.200V0)
Sterile 4-mm diameter glass beads (Dutscher, catalog number: 068502)
Sterile 96-well V-bottom clear polystyrene microplates (Greiner Bio-One, catalog number: 651101)
Sterile pipette tips
Compost (Proveen, catalog number: 14926)
Cauliflower: Brassica oleracea var. botrytis cv. Clovis F1 (Vilmorin)
Note: Plants are grown under greenhouse conditions and used at 4 weeks after sowing. All inoculations were performed using the second true leaf (second leaf above the cotyledons). After inoculation, plants are kept in classical growth chamber conditions: 9 h light, 22°C, and 70% relative humidity.
Xanthomonas campestris pv. campestris (Xcc) strain 8004::GUS-GFP (WT hereafter; Cerutti et al., 2017) and variants: Xcc 8004::GUS*-GFP* (WT* hereafter, carrying the point mutations Y468A and Y66A inactivating the catalytic sites of the β-glucuronidase and GFP proteins respectively); Xcc 8004::GUS-GFP ΔhrpX; Xcc 8004::GUS-GFP ΔhrcV; Xcc 8004::GUS-GFP ΔxopAC
Note: With concerns to the cost of fitness related to the constitutive expression of the GUS-GFP cassette, we decided to create the non-functional version of the cassette for our reference strain. With this system, the two challenged strains carry and express the GUS-GFP cassette, but only one of them will actually appear as tagged. We thus remove the possibility of a fitness cost associated with the cassette while still being able to differentiate the two mixed strains.
Spectrophotometer cells (Standard Polystyrene Semi-micro Cuvettes, Sarstedt, catalog number: 67.742)
Sterile distilled water
Magnesium chloride (MgCl₂) (Merck, catalog number: 105832)
Yeast extract (Sigma-Aldrich, catalog number: Y1625)
Casamino acids (BD, BD Biosciences, catalog number: 223050)
Potassium phosphate dibasic (K₂HPO₄) (Merck, catalog number: 105101)
Agar (BD, BD Biosciences, catalog number: 214010)
Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Merck, catalog number: 105886)
X-Gluc (5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid, cyclohexylammonium salt monohydrate) (BIOSYNTH, catalog number: B-7300)
DMF (N,N Dimethylformamide) (Sigma-Aldrich, catalog number: D4551)
Rifampicin (Euromedex, catalog number: 1059)
Methanol (VWR, catalog number: 20847.320)
Pimaricin (DSM, DEVLOCID Instant, catalog number: 00226)
MOKA medium (see Recipes)
1 mM MgCl₂ solution (see Recipes)
X-gluc (see Recipes)
Rifampicin (see Recipes)
Pimaricin (see Recipes)

Equipment

6-mm diameter Hollow punch (Harris, Uni-Core, Electron Microscopy Sciences, catalog number: 69039-60)
Hand-held colony counter (Heathrow Scientific, catalog number: HS120000)
28°C bacteria incubator
Centrifuge (Eppendorf, model: 5415D)
Beads-assisted grinder (Retsch, catalog number: MM400)
Spectrophotometer (Amersham Biosciences, model: Ultrospec 2100 pro)
Sterile environment (bunsen burner or microbiology hood)
Multi-channel pipette (10–100 µL)
Mono-channel pipettes

Procedure

Preparation of bacterial cells for the inoculation
1. Bacterial cells grown on MOKA plates with appropriate antibiotics (i.e., Rifampicin 50 µg/mL here) are used to inoculate 5 mL of liquid MOKA medium (with same antibiotics) and grown overnight at 28°C under agitation at 200 rpm.
2. Centrifuge 2 mL of bacterial preculture for 5 min at 6,000 rpm (i.e., 3.3 × g) and wash twice in 1 mM MgCl₂. Resuspend in 1 mM MgCl₂ and measure OD_600nm.
1. Prepare 1 mL of bacterial suspension in a 2-mL tube so that, for each strain, final OD_600nm = 0.1 (i.e., 1 × 10⁸ cfu/mL; note: this estimation has to be verified when working with other species by plating on solid medium) in 1 mM MgCl₂.
2. Dilute each suspension to 1/100 in a 5-mL tube to obtain a final inoculum at OD_600nm = 0.001 (i.e., 1 × 10⁶ cfu/mL) in 5 mL of 1 mM MgCl₂. For the WT* reference strain, prepare 10 mL of suspension at OD_600nm = 0.001 as it will be used in all mixes.
3. Mix 2 mL of WT* suspension with 2 mL of each of the strains to test in another 5-mL tube (so that each mixed strain is at 5 × 10⁵ cfu/mL, but the final concentration of the inoculum is 1 × 10⁶ cfu/mL).
Inoculation
1. Using a blunt syringe filled with one milliliter of bacterial suspension, infiltrate the mesophyll of the 2^nd true leaf of cauliflower (for pictures of the procedure, see Cohn et al., 2015). A large enough area should be infiltrated in order to later be able to sample a 6-mm leaf disc without taking any of the tissues damaged by the syringe. Typically, up to seven separate spots can be infiltrated on the same cauliflower leaf, thus allowing the inoculation of multiple strains or mixes on the same leaf. In this case, be careful to well separate infiltration areas to exclude cross-contamination.
2. Circle the infiltrated area on the lower side of the leaf to mark the infected tissue for later sampling.
3. Place the plants in the growth chamber and water them appropriately.
Plating of the inoculation suspensions and counting of the initial relative frequencies in mixed populations
1. Transfer 100 µL of inoculation suspension into a 96-well plate with conical bottom.
2. Perform serial 1/10 dilutions (10 µL in 90 µL of 1 mM MgCl₂) using a multichannel pipette.
3. Take 100 µL of dilution 10^-3 and spread out them on round 92-mm MOKA plates containing the appropriate antibiotics (i.e., Rifampicin 50 µg/mL here) and fungicide (i.e., Pimaricin 30 µg/mL here) as well as X-Gluc 200 µM. In our case, spreading is performed by shaking using 4-mm glass beads until homogeneous repartition of the suspension.
4. Incubate 4–5 days at 28°C to allow bacterial growth and blue staining of GUS-expressing colonies.
5. Count blue and white colonies, e.g., using a hand-held colony counter, to determine the ratio of mixed strains at T₀. This is necessary for proper normalization during later calculation of the CI (see Data analysis section).
Sampling, plating, and counting of relative frequencies in mixed populations after incubation
1. Three days after inoculation, sample mesophyll tissues by punching out a 6-mm leaf disc in the infiltrated area defined by the marker label and placing it in 200 µL of 1 mM MgCl₂.
2. Grind leaf samples using 4-mm glass beads in 2-mL tubes (2 beads per tube) using the beads-assisted grinder for 2 min at 30 Hz.
3. Centrifuge shortly (5 s) the tubes containing the ground material to gather it at the bottom.
4. Transfer 100 µL of ground tissue to a 96-well plate with conical bottom.
5. Proceed as described in Steps C2 to C5. However, because each strain displays a different phenotype, and to ensure the presence of colonies from both strains, plate 50 µL of 10^-2 and 10^-3 dilutions.
Note: Classically, we advise collecting bacterial titers by counting the plates displaying between 30 and 300 colonies. However, because competition assays require to finely determine the number of CFU for each of the mixed strains, we recommend counting plates where there are at least 100 colonies for mix-inoculated samples.

Data analysis

For reproducibility purposes, experiments should be performed in at least four biological replicates. A biological replicate is defined as a replication of a complete experimental procedure with fresh buffers, fresh bacterial cells suspension prepared from cells grown on new solid medium, and new batches of plants. Results of competition assays are displayed as a Competitive Index (CI), which is defined as the ratio of the relative abundance of mutant over WT* strain after incubation normalized by the relative abundance of mutant over WT* strain in the inoculum (Taylor et al., 1987; Macho et al., 2007):

N: number of colonies

dpi: days post inoculation

Here, we used the Xcc 8004::GUS-GFP ΔhrpX, ΔhrcV, and ΔxopAC mutants as an example for this protocol. These three strains are Type III Secretion System-related mutants that exhibit contrasted fitness in planta (Feng et al., 2012; Guy et al., 2013; Wang et al., 2015). Competitive indices of these three strains, when co-inoculated with the WT* strain, are shown in Figure 1. Xcc 8004::GUS-GFP is a negative control whose fitness should be the same as the WT* reference strain (i.e., CI ~1). Strains with CI < 1 are considered less fit than the WT strain in the mesophyll environment.

We used four independent biological replicates of one plant sample each. The plot was generated using the ggplot2 package of the R software (Wickham, 2016; https://github.com/tidyverse/ggplot2). We purposely set a log10-based scale to stretch out low CI values that would be hardly differentiable otherwise. Statistical treatment of the data was also achieved with R using the Kruskal-Wallis test. Groups identified as significantly different (P < 0.05) are displayed using the letters ‘a’, ‘b’, and ‘c’ in Figure 1A.

Figure 1. Competition assay of Xcc 8004::GUS-GFP strain and related mutants in cauliflower mesophyll. All competition assays were performed by co-inoculating the Xcc 8004::GUS*-GFP* reference strain with Xcc 8004::GUS-GFP derivatives in a 1:1 ratio. Mesophyll discs were sampled at 3 days post-inoculation. A. Graphical representation of competition assays results. Strains showing CI ~1 are considered as fit as the WT strain, while strains with CI < 1 are considered less fit than the WT strain in the mesophyll environment. Horizontal bars show the median for four independent biological replicates of one plant sample each. Statistical significance of fitness differences was evaluated by a Kruskal-Wallis test. Groups identified as significantly different (P < 0.05) are displayed using the letters ‘a’, ‘b’, and ‘c’. Results showing the same letter belong to the same statistical group. B. Photo of a plate displaying colonies from the WT vs. WT* sample at 3 dpi in the mesophyll. Visually, the two populations are roughly present in a 1:1 ratio, illustrating the expected equivalent fitness of both strains. The same 1:1 ratio is obtained after plating any of the inoculation suspensions. C. Photo of a plate displaying colonies from the ΔhrpX vs. WT* sample at 3 dpi in the mesophyll. Blue-stained colonies of the ΔhrpX strain are strongly under-represented, clearly exposing the large deficit of fitness of this strain.

Recipes

MOKA medium
1. Mix 4 g of yeast extract, 8 g of casamino acids, 2 g of K₂HPO₄, and 0.3 g of MgSO₄·7H₂O.
2. Add distilled water to 1 L.
3. Mix until complete dissolution of the reagents.
4. Sterilize 250-mL aliquots of the mixture by autoclaving at 120°C for 20 min.
5. Store at room temperature.
6. Solid MOKA medium for plates is prepared identically and complemented with 3.75 g of agar added to each individual 250-mL aliquot before autoclaving.
1 mM MgCl₂ solution
1. Prepare a stock solution of 1 M MgCl₂ by dissolving 2.033 g of MgCl₂·6H₂O in 10 mL of dH₂O.
2. Sterilize by autoclaving at 120°C for 20 min.
3. Prepare 1 mM MgCl₂ by diluting the stock solution 1/1,000 in sterile dH₂O.
4. Both solutions are stored at room temperature.
X-gluc
1. Prepare X-gluc 500x stock solution by dissolving 0.54 g of X-gluc in 10 mL of DMF.
2. Store at -20°C.
3. Add 500 µL of X-gluc stock solution to 250 mL of MOKA + Agar solution to get a final concentration of 200 µM of X-gluc in plates used for CI measurement.
Rifampicin
1. Dissolve rifampicin powder in methanol to obtain a stock solution at 10 mg/mL.
2. Store at -20°C.
3. Dilute rifampicin stock solution to 1/200 in MOKA media to obtain a final concentration of 50 µg/mL.
Pimaricin
1. Resuspend pimaricin powder in water to obtain a stock solution at 30 mg/mL.
2. Store at 4°C.
3. Dilute pimaricin stock solution to 1/1,000 in MOKA media to obtain a final concentration of 30 µg/mL.

Acknowledgments

This work was supported by a Ph.D. grant from the French Ministry of National Education and Research to JSL. JSL, LDN, EL, and AB are supported by the ANR (ANR-19-CE20-0014-01). LIPM is part of the French Laboratory of Excellence project (TULIP ANR-10-LABX-41; ANR-11-IDEX-0002-02).

Competing interests

There are no competing interests.

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