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+## SynCom invasion experiments
+
+FlowPots were sequentially inoculated with native and non-native strains. FlowPots were prepared as usual, with the addition of a round nylon filter (pore size 200 µm) at the bottom of the pot to avoid clogging of the bottom opening by matrix material. FlowPots were first inoculated with the mixed SynCom (16 *Lj*- and 16 *At*-strains), the *At* SynCom (16 *At*-strains), the *Lj* SynCom (16 *Lj* strains) or the mock solution (medium only). The medium used for inoculation was 0.25× B&D (Broughton, W. J. & Dilworth, M. J., 1971) supplemented with 1 mM KNO3 for both plant species.
+
+For sterilization, *At* seeds were incubated for 5 min in 70% ethanol, then twice for 1 min in 100% ethanol, washed five times with sterile water and stored at 4 °C in the dark for stratification. *Lj* seeds were scarified by abrading the surface using sand paper, incubated for 20 min in diluted bleach and washed five times with sterile water. Sterilized seeds were placed on sterile Whatman paper wetted with sterile water in a squared petri dish and allowed to germinate under short-day conditions. Sterilized Col-0 seeds and germinated sterile Gifu seeds were placed on the soil surface. Note that a few drops of *Mesorhizobium* culture (*Lotus* root nodule symbiont, strain LjNodule218, OD600 0.02) were applied to Gifu seedlings in the *At* SynCom treatment to allow for normal root nodule symbiosis to occur and ensure healthy plant growth. After growth for 4 weeks, a second inoculation was performed, where a mock inoculum (medium) was added to the mixed SynCom-treated pots, the *Lj* SynCom was added to the *At* SynCom-treated pots, the *At* SynCom was added to the *Lj* SynCom-treated pots and mock inoculum was added to the mock-treated pots. The pots were flushed in reverse by adding the inoculum from the top and applying vacuum from the bottom. On a sterile bench, FlowPots (cut 60-ml syringes with a male Luer Lok connector) were placed onto female Luer Lok connectors of a vacuum manifold (QIAvac 24 Plus, Qiagen), keeping the valves of the manifold closed. Vacuum was applied to the manifold with an attached vacuum pump. Next, 20 ml of inoculum were carefully added to a pot with a 20-ml syringe and needle, avoiding damage of the plant shoots. Each pot was inoculated by opening and closing the corresponding valve. Pots were put back into the plastic containers and plants grown for another 2 weeks. Root, rhizosphere and soil samples were harvested as described above.
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