Culturing and Growth Conditions of Fungi and Arabidopsis

Twelve-day-old Arabidopsis (Arabidopsis thaliana) seedlings were transferred from Murashige and Skoog plates to plates with solid plant nutrition medium and a nylon membrane (mesh size, 70 µm) as described previously (Camehl et al., 2011). Seedlings were then grown for an additional 24 h under long-day conditions, with light applied from above (60–70 µmol m⁻² s⁻¹, 16 h of light/8 h of dark), at 22°C. These seedlings were then used for the different experiments: transfer to fresh plates with fungi or incubation of the roots with elicitors.

For elicitor application, the roots were soaked in a solution containing either CT or other chemicals (see below), and autoclaved water was used as a control. The plates with the seedlings were then transferred back to long-day conditions, before the roots were harvested after the treatments described in the text.

Transgenic Arabidopsis expressing cytosolic apoaequorin in the Col-0 background (pMAQ2) was a gift from Marc Knight (Knight et al., 1991; Polisensky and Braam, 1996), rbohd knockout seeds were a gift from Jonathan D.G. Jones, and tpc1 in the aequorin background was a gift from Edgar Peiter. Knockout lines for GRL2.4 (SALK_010571C), GRL2.5 (SALK_078407C), and GRL3.3 (SALK_099757C) were obtained from TAIR. After crossing with pMAQ2, homozygote knockout lines were generated using the primer pairs given by TAIR. Ethyl methanesulfonate mutants of pMAQ2 seeds, which were screened here, were generated in an earlier study (Johnson et al., 2014).

Piriformospora indica was cultured and maintained on Kaefer medium, pH 6.5, as described by Johnson et al. (2011b). The fungus also was grown on Kaefer medium broth for 18 d at 22°C to 24°C in complete darkness on a horizontal rotating shaker at 50 rpm for the preparation of the cell wall extracts (Vadassery and Oelmüller, 2009; Johnson et al., 2011b).

Alternaria brassicae (FSU-3951), Mortierella hyalina (FSU-509), and Verticillium dahliae (FSU-343) were obtained from the Jena Microbial Resource Centre, and Periconia macrospinosa was from Gabor Kovács. The fungi were grown on potato dextrose agar medium (pH 6.5–6.7) at 20°C ± 1°C in a temperature-controlled chamber under 12/12 h of light/dark and 75% relative humidity for 2 weeks. The fungi were inoculated to Arabidopsis seedlings and reisolated from the infected tissues every 6 months (Johnson et al., 2013). Agrobacterium tumefaciens was grown for 3 d in a medium containing 1% (w/v) yeast extract, 1% (w/v) tryptone peptone, and 0.5% (w/v) NaCl (pH 7) at 27°C, with shaking at about 200 rpm.