A1296

Wild type (WT) plants used in this study were Columbia (Col-0) background in Arabidopsis and the WT were fielder in Triticum aestivum. The T-DNA insertion lines nramp1 (SALK_053236C) were obtained from Nottingham Arabidopsis Stock Centre (NASC).
Arabidopsis seeds were grown on a nutrient medium consisting of 1% agar (Sigma-Aldrich; A1296; USA), 1% sucrose, and full-strength Hoagland nutrient solution (5 mM KNO₃, 5 mM Ca(NO₃)₂, 2 mM MgSO₄, 1 mM NH₄H₂PO₄, 20 μM MnSO₄, 3 μM H₃BO₃, 1 μM (NH₄)₆Mo₇O₂₄, 0.4 μM ZnSO₄, 0.2 μM CuSO₄, 20 μM Fe (III)-EDTA) at pH 5.75 ~ 5.85, and -Mn medium (full strength Hoagland nutrient solution without MnSO₄), grown on vertical plates at 21 °C for 10 days, 16 h light/8 h dark cycle (Gao et al. 2018 ). The medium for Fe deficiency and Zn deficiency referred to the previously published methods, and some adjustments were made (changed 1/20 Hoagland to 1 × Hoagland) (Wang et al. 2023 ; Gao et al. 2018 ).
For phenotyping assay in Triticum aestivum, WT, TaNRAMP3-OE and TaNRAMP3-RNAi plants were grown hydroponically in Hoagland medium for one week and then transferred to Mn-deficiency medium for another two weeks. All phenotypic experiments were repeated three times (n = 15 for each genotype). The root length was measured with ImageJ.

For the purpose of this study, the fungal isolate was plated from glycerol stock and grown on nutrient-poor malt agar with sea salts [4 g/L malt extract (Moss Malt Extrakt, Jensen & Co AS), 40 g/L sea salts (S9883, Sigma-Aldrich), 15 g/L agar (A1296, Sigma-Aldrich) and Milli-Q^® H₂O] until the growth covered the entire agar plate (approximately 40 days). Milli-Q^® H₂O was produced with the in-house Milli-Q^® system. One-half of the agar plate covered in mycelium was used to inoculate each liquid culture, in malt medium with added sea salts (4 g/L malt extract, 40 g/L sea salts). Two cultures of 200 ml were inoculated and incubated for 107 days at static conditions and 13°C. Before the addition of resin for extraction, mycelium was taken from the culture for inoculation of another round of cultures. The second cultivation contained four cultures with 250 ml of malt extract medium supplemented with sea salts and cultivated under the same conditions for 83 days. The total culture volume used for the extraction of 1 was 1.4 L. The cultures were extracted using Diaion HP-20 resin (13607, Supelco) and methanol (20864, HPLC grade, VWR) as described previously (Kristoffersen et al., 2018 (link); Schneider et al., 2020 (link)). The extract was dried in a rotary evaporator at 40°C under reduced pressure and stored at −20°C.

600 μl of 0.6% agar (Sigma, A1296) was added to each well of a 24-well plate. After the 0.6% agar solidified, the digested cells mixed with 0.3% agar were inoculated into the same at 600 μl/well; each well contained 1000 cells. The colonies that were >50 μm in diameter were photographed and counted under a stereomicroscope (Olympus, Japan) after three weeks.

Mix the cells (5 × 10²) and 2 mL complete medium along with 0.3% agar (Sigma, A1296) to an agar-cell mixture, seed this agar-cell mixture on the top of a bottom layer of 1% complete medium agar, and culture for 10 days. Viable colonies that contained >50 cells or were >0.1 mm were counted. Colony size was measured with an ocular micrometer. All experiments were performed in triplicates.

Semi-solid culture conditions were set up using culture media supplemented with agar^{54 (link)}. Briefly, prewarmed DMEM-F12 supplemented with 20% FBS and 1% penicillin–streptomycin (complete medium) was mixed 1:10 with 2.5% (w/w) agar (#A1296; Sigma) dissolved in sterile water. Two milliliters of media/agar mix was added to each well of a six-well-plate and allowed to set at room temperature for 30 min, before tissue was placed on top and the dish was covered with complete medium.