Agar

Mueller-Hinton (Beckton-Dickinson, Sparks, MD, USA) and LB-plates (Merck, Darmstadt, Germany) were prepared with 13 g/l agar (Merck), and RH-plates were made by autoclaving 100 ml milliQ water with 7.5 g agar, cooling it to 55°C and mixing with 500 ml modified RPMI-medium (Sigma, R7388) prewarmed to 55°C. +U, +C, and +Mg, indicates addition of 20 mg/l uracil, 10 mg/l chloramphenicol, and 5 mM MgCl₂, respectively.

Sabouraud broth containing 0.5% Witte’s peptone, 2% glucose and 1.8% agar ((Sigma-Aldrich, Budapest, Hungary), in distilled water was autoclaved at 121 °C for 20 min at pH 6.0 and was used to prepare agar slant tubes. Streaking to plate out cells across the surface of the agar slants took place with the sterile loop of the wire inoculator containing a drop of cell culture. Fungal cells were incubated for six days at 34 °C. Spores were removed with 50 mL physiological salt solution (saline) and completed with saline to 100 mL, shaken for 24 h at 28 °C to get homogeneous distribution. After counting the living cell number (e.g., Fusarium), cell suspensions were stored in a refrigerator at 5 °C until further use. For infection, the initial germ number of the suspension for Fusarium and Trichophyton species was diluted to 10⁵ spores/mL, for Microsporum gypseum 10⁶ spores/mL. For the other fungi, including the yeast Candida species, the initial cell number was set to 2 × 10⁴ cells/mL. In vitro incubations in duplicate samples lasted for six days at 34 °C.

Sheep red blood cells (SRBCs) were purchased from South Pacific Sera Co. (Timaru, New Zealand), and Earle’s balanced salt solution (EBSS), DEAE-dextran, agar, 2-mercaptoethanol, guinea pig complement, agar, and cyclophosphamide (CY) reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Roswell park memorial institute medium (RPMI1640), fetal bovine serum (FBS), penicillin–streptomycin, L-glutamine, and hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer were purchased from Gibco Co. (Rockville, MD, USA), and ammonium-chloride-potassium (ACK) lysis buffer was purchased from Lonza Co. (Walkersville, MD, USA). Antibodies, including purified anti-mouse CD16/CD32 Fc receptor, peridinin chlorophyll-a protein (PerCP)-conjugated anti-mouse CD3e (clone: 145-2C11), R-phycoerythrin (R-PE)-conjugated anti-mouse CD45R/B220 (clone: RA3-6B2), and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD11b (clone: M1/70) were purchased from BD Pharmingen Inc. (San Diego, CA, USA), and the phagocytosis assay (IgG FITC) kit was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA).

Ceratopteris richardii strain Hn-n^{40 (link)} was used in this study to generate the transgenic plants. Gametophytes were grown on FM plates (pH 6.0) containing 0.5 × MS salts (PhytoTechnology Laboratories) and 0.7% (w/v) agar (Sigma-Aldrich). Sporophytes were formed on fertilized gametophytes and were transferred to soil, typically after 3–4 weeks of fertilization. Both gametophytes and young sporophytes were grown under continuous light at 28 °C. Adult sporophytes were grown in the LILY greenhouse facility at Purdue for harvesting spores.
Ceratopteris calli were induced from young sporophytes (shoot tips or fronds) on the callus induction medium (pH 5.8) that contains 1 × MS salts (PhytoTechnology Laboratories), 2% (w/v) sucrose, 1 mg/L benzylaminopurine (BAP), and 0.7% agar (Sigma–Aldrich). Calli were cultivated under continuous light at 28 °C in the growth chamber (Percival).

For mannitol (Sigma-Aldrich, Saint Louis, MO, USA) treatment, seeds were plated on 1/2 MS medium, pH 5.7, containing 1% agar, vertical cultured for 4 days, and transferred to 1/2 MS medium containing 0, 100, 200, and 300 mM mannitol; the position of root tips was marked. Root length was measured after 3 days of growth. For PEG treatment, PEG (PEG8000, Sigma-Aldrich, Saint Louis, MO, USA)-infiltrated plates were prepared according to Verslues et al. [8 (link)] using 10 × 10 square Petri dishes and the PEG-infused plates were allowed to rest for 12 hours before use. The water potential of the plates was measured with a Dewpoint Potential Meter (WP4C, Pullman, WA, USA). Seeds were vertically grown in 1/2 MS medium (pH 5.7) containing 6 mM MES (Sigma-Aldrich, Saint Louis, MO, USA) and 1.5% agar (Sigma-Aldrich, Saint Louis, MO, USA) for 4 days, transferred to PEG-infiltrated plates; the position of root tips was marked and grown for 3 days. The plates were photographed, and root length from the marked position to the new position of the root tip was measured with ImageJ 1.48v software (National Institutes of Health, USA).