Potato dextrose broth (pdb)

Verticillium strains (Supplementary Table S7) were cultivated in liquid SXM modified from Neumann and Dobinson (2003) (link) as described by Hollensteiner et al. (2017) (link) for conidiospore formation and in liquid potato dextrose medium [Potato Dextrose broth (Carl Roth)] for mycelial growth. Cultures were incubated at 25°C under constant agitation at 115−125 rpm. For long-term storage spores were maintained in closed vials with 25% glycerol at −80°C.
For the exoproteome comparison, V. longisporum 43 (Vl43) was inoculated with 1.5 × 10⁶ spores per 150 ml potato dextrose medium and incubated for 4 days at 25°C and 150 rpm agitation. Each culture was centrifuged and the mycelium and spore sediment was resuspended in 150 ml extracted xylem sap of B. napus; SXM, the minimal medium Czapek-Dox medium (CDM, modified from Smith, 1949 (link)) supplemented with 7% extracted xylem sap or plant proteins; H₂O and H₂O supplemented with 0.1% glucose; YNB (yeast nitrogen base: 1.5 g/l YNB, 5 g/l (NH₄)₂SO4, 20 g/l glucose, ad 1 l H₂O) and 10% vegetable juice (V8, Campbell Soup Company, Sexton et al., 2009 (link)). After an additional incubation period of 4 days, proteins of the supernatant were precipitated with TCA/acetone.

The identified fungal species B. cinerea was cultivated on PDA (Carl Roth) with 2% micro agar (Duchefa). Stock cultures of fungal mycelia were transferred to fresh PDA plates bi-weekly. To collect fungal mycelium, B. cinerea was grown for 7 days at 23 °C in darkness. To obtain spores, the fungus was grown in darkness at 28 °C for eight days [31 (link)]. For the harvest of spores, 1.5 mL 10% sterile glycerol was pipetted on the culture plate, and the spores were gently scraped with a sterile plate spreader (TH Geyer, Höxter, Germany). Spores in glycerol were stored at − 80 °C.
Liquid cultures of B. cinerea were produced by inoculating 100 mL Potato-Dextrose-Broth (Carl Roth) in 500 mL Erlenmeyer flasks with five 6 mm fungal agar plugs punched out with a cork borer from a seven-day-old PDA plate with B. cinerea mycelium. The cultures were grown for 7 days on a shaker at 120 rpm at 23 °C in darkness.

For P. arabidopsidicola cell wall isolation, cells were cultivated in potato dextrose broth (PDB; Carl Roth) in 200 ml flasks on a rotary shaker for 4 days at room temperature. Cells were collected and washed twice in sterile MQ water, re-suspended in a small volume of sterile MQ water, and freeze-dried for 2 days. Dry cells were ground in a tissue homogenizer (Precellys 24)⁵ using 425–600 μm glass beads (5×15 s at 6,800 rpm, cooling at-20°C between runs). Cell disruption was confirmed using a compound microscope (Leica Model MZ 2500)⁶ with 40× objective. The insoluble cell walls were separated from the soluble fraction by centrifugation in MQ water at 1,000 g for 20 min in + 4°C. This crude cell wall preparation was then freeze dried as described above, and weighed.
Sterile Arabidopsis Col-0 accession seeds were stratified at + 4°C for 72 h and germinated vertically on 0.5× MS 0.8% agar plates for 4 days in a growth chamber (Fitotron SGC120, Weiss Technik)⁷ at ~170 μmol m⁻² s⁻¹ illumination, at +23/+18°C. Seedlings were transplanted to either 0.5× MS agar control plates or identical plates supplemented with 0.9 g/l P. arabidopsidicola cell wall preparation. Seedlings were grown in the same conditions for 10 more days, photographed, and root length measured using imageJ software.⁸

Pure cultures of A. flavus, A. parasiticus, A. nidulans, A. niger, Fusarium oxysporum, F. culmorum, Phytophthora nicotianae, Rhizoctonia solani, Pythium ultimum, Botrytis cinerea, Cercospora nicotianae, Thielaviopsis basicola, and Penicillium chrysogenum were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and maintained on potato dextrose agar (PDA; Carl Roth, Karlsruhe, Germany), tomato agar (25% (v/v) tomato juice, 3 g/l CaCO₃, 15 g/l agar) or liquid potato dextrose broth medium (PDB; Carl Roth). The wild-type A. fumigatus strain D141 (Reichard et al., 1990 (link)) and Galf-deficient mutant ΔglfA were cultivated as previously described (Schmalhorst et al., 2008 (link)). The F. oxysporum strain DSM 62316 used for immunofluorescence analysis and enzyme-linked immunosorbent assay (ELISA) experiments was cultivated and prepared as previously reported (Wiedemann et al., 2016 (link)).

Serendipita (=Piriformospora) indica strain DSM 11,827 was provided by Pr. Philipp Franken and obtained from the “Deutsche Sammlung für Mikroorganismen und Zellkulturen,” Braunschweig, Germany (Varma et al., 1999 (link)). The fungus was maintained at −80°C in sterile Potato Dextrose Broth (PDB) (Carl Roth, Germany) amended with 25% glycerol and grown on Potato Dextrose Agar (PDA) plates or in liquid culture containing Aspergillus complete medium (Pontecorvo et al., 1953 (link)).
To produce inoculum, roughly fifty 2-mm agar plugs from a 2-week old culture of S. indica grown on PDA were transferred to 250 ml Erlenmeyer flasks containing 100 ml of Aspergillus CM and incubated for 3 weeks under constant shaking (150 rpm) at 26 ± 1°C. Mycelium and spores were collected by centrifugation (4,500 rpm, 5 min) and the remaining pellet was washed 3–5 times with sterile phosphate-buffered saline (PBS) of pH 6.5. The mixture of mycelium and spores, resuspended in PBS, was ground with a homogenizer Ultra Turrax T25 (IKA^®, Staufen, Germany) for 3 min in intervals of 30 s. The number of spores + mycelium fragments was estimated with a hemocytometer (NanoEnTek, Seoul, Korea) and the viability of the CFU confirmed by plating on PDA. Final concentrations were adjusted with PBS.