Gellan gum

The D. discoideum KAx3 strain was grown in association with Klebsiella aerogenes on 1/2 SM agar plates (20.9 g/L SM agar (Formedium), 11.25 g/L KH₂PO₄, 3.4 g/L K₂HPO₄, and 8.5 g/L agar) or in HL-5 liquid axenic medium (Formediun) with 150 rpm rotation at 22°C. The cells were harvested at late log stage (we call these condition of plates as “half clear”) and washed with KK2 buffer (16.5 mM KH₂PO₄, 3.9 mM K₂HPO₄ pH6.2) at least twice to eliminate bacteria or HL-5 medium completely. Washed cells of D. discoideum were plated on KK2 buffered 1.5% phytagel or gellan gum (Sigma) in 6 cm φ petri dishes (referred to as tester plates) directly or on filter paper at the density of 1 x 10⁸ cells /cm². M. incognita second-stage juveniles were prepared as described previously [11 (link)].
D. purpureum was a kind gift from Dr R. Kay (MRC Laboratory of Molecular Biology, UK) and Polysphondylium pallidum PN500 was a kind gift from Dr P. Schaap (University of Dundee, UK). D. fasciculatum S350 was provided by NBRP-nenkin (http://nenkin.nbrp.jp). These species were grown in association with Escherichia coli B/r on 2–5 LP agar plate. LP agar plate was made according to the previous report with modification [12 (link)]. In brief, we used lactose instead of glucose for LP plate.

Seeds of Arabidopsis accessions and T-DNA mutants were purchased from ABRC (Arabidopsis Biological Resource Center). Seeds were surface-sterilized with 5% (v/v) sodium hypochlorite and 0.05% (v/v) Tween 20 for 5 min and were placed on half strength Murashige and Skoog medium (0.23% (w/v) MS salts (Fujifilm Wako Pure Chemical, Osaka, Japan), 0.5% (w/v) sucrose, and 0.03% (w/v) gellan gum (Sigma-Aldrich, MO, USA), pH 5.8). After seeds for sowing were stored at 4 °C for 2 days in the dark, plates were placed in a vertical position under continuous light conditions at 22 °C to allow the seedlings to grow onto the surface of the medium.

The embryo sacs were cut near the suspensor and initially cultured for 3 months at 23 °C in darkness on ESM induction medium. The ESM induction medium consisted of DCR medium [49 (link)], supplemented with 2–10 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg/L 6-benzyladenine (6-BA), 0.5 mg/L Kinetin (KT), 20 g/L maltose, 450 mg/L glutamine, 500 mg/L casein hydrolysate (CH) (Sigma, St. Louis, MO, USA), 100 mg/L inositol and 2.4 g/L gellan gum (Sigma, St. Louis, MO, USA). The pH of the medium was adjusted to 5.8 ± 0.1 with NaOH or HCl, after which it was autoclaved at 121 °C for 20 min.

Dual-culturing of S. elegans and R. solani was conducted in 9 cm Petri plates containing 20 mL of minimal synthetic medium (MSMA) composed (g L⁻¹) of: MgSO₄.7H₂O, 0.2; K₂HPO₄, 0.9; KCl, 0.2; FeSO_4.7H₂O, 0.002; MnSO₄, 0.002; ZnSO₄, 0.002; NaNO₃, 1.0; biotin, 10 mg; gellan gum, 1% (composed of glucose, glucuronic acid and rhamnose in the molar ratio of 2:1:1) (Phytagel, Sigma, St. Louis, USA).
Agar plugs (8 mm) of a 5-day old R. solani culture were grown on MSMA for 48 h and then sprayed with 100 μL of a suspension of S. elegans conidia (10⁶ mL⁻¹ water) using a Badger 350 air brush and MC-80 mini air compressor calibrated at 1 kg cm⁻². The control treatments consisted of spraying 100 μL of S. elegans conidia on non-inoculated MSMA plates and R. solani-inoculated MSMA plates sprayed with sterile distilled water. Additionally, a negative control representing the MSMA medium was used to determine compounds of non-biological origin. All culture plates were incubated at 24°C for 4 or 5 days following dual and pure strain cultivation. These time points were chosen based on a priori knowledge to capture the infection and colonization of R. solani hyphal cells by S. elegans (Chamoun and Jabaji, 2011 (link)). Five replications were performed per treatment.

Gellan gum (Gelzan), glycerol (Gly), methacrylic anhydride (MAA), triethylamine (TEA), anhydrous dimethyl sulfoxide (DMSO), deuterated water (D₂O), 4-dimethylaminopyridine (4-DMAP), phosphate buffer saline (10 mM phosphate and 150 mM NaCl, pH = 7.4, which was labelled PBS), polyethylenglycole (PEG 20,000 Da), 2-hydroxy-4-(2-hydroxyethoxy)-2-methyl-propiophenone (Irgacure 2959) and dialysis tubes (cut-off 12–14 kDa) were purchased from Sigma Aldrich (Milan, Italy). Silver nitrate (AgNO₃), monobasic potassium phosphate (KH₂PO₄, used to prepare 40 mM phosphate buffer, pH = 7.4, which was labelled PB), sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from Carlo Erba (Milan, Italy). Finally, 1-methyl-2-pyrrolidinone from Fluka (St. Louis, MO, USA). Triptone Soy Agar (TSA) and Brain Heart Infusion broth (BHI) were purchased from Oxoid (Basingstoke, UK).