Growth medium and conditions for seedlings and fungi
Seeds of wild-type A. thaliana (ecotype Columbia-0), and slim1(Maruyama-Nakashitaet al. , 2006) mutant were surface-sterilized for 8 mins in sterilization solution containing lauryl sarcosine (1%) and Clorix cleaner (23%). Surface-sterilized seeds were washed with sterilized water 8 times and placed on Petri dishes with MS medium supplemented with 0.3% gelrite (Murashige and Skoog, 1962). The MS medium contains 1.5 mM MgSO4. After cold treatment at 4 °C for 48 - 72 hours, plates were incubated at 22 °C under long day conditions (16 hours light/ 8 hours dark; 80 μmol m−2 s−1).
Sulfur deficiency assays were performed with MGRL medium (Fujiwaraet al. , 1992). 1 L of MGRL medium contains 1.75 mM NaH2PO4, 1.75 mM Na2HPO4, 2 mM Ca(NO3)2, 1.5 mM MgSO4, 3 mM KNO3, 67 µM Na2EDTA, 30 µM H3BO3, 10.3 µM MnSO4, 8.6 µM FeSO4, 1 µM ZnSO4, 1.0 µM CuSO4, 130 nM CoCl2, 24 nM (NH4)6Mo7O24, 1% sucrose, 0.3% Gelrite, pH 5.6. For MGRL medium with reduced sulfate, MgSO4 was replaced by MgCl2. The total sulfate concentration in high (HS) and low (LS) sulfate MGRL medium is 1520.9 µM and 20.9 µM, respectively.
Mortierella strains (M. hyalina , FSU-509; M. alpina , SF002698; M. turficola , SF009851; M. vinacea , SF002701; M. longicollis , SF009830) were obtained from Jena Microbial Resource Center (Jena, Germany). They were grown on Potato-Dextrose-Agar (PDA), pH 6.5, and at 23 °C in the dark (Bains and Tewari, 1987) for fresh subcultures and desiccator assays.
For the sulfur labeling assays, M. hyalina was grown on KM medium modified from Hill and Käfer (2001): 1 L of the medium contains 7.06 mM NaNO3, 6.98 mM KCl, 11.17 mM KH2PO4, 177.9 μM H3BO3, 6.4 μM CuCl2, 76.5 μM ZnCl2, 7.28 μM CoCl2, 0.89 μM (NH4)6Mo7O24, 29.6 μM MnCl2, 20 μM Na2EDTA, 20 μM FeCl2, 2% glucose, 0.2% peptone/trypton, 0.1% yeast extract, 0.1% casein hydrolysate, 1% agar. Finally, 2.11 mM ammonium sulfate ((NH4)232SO4or (NH4)234SO4) was added to make the unlabeled (32S)/ labeled (34S) medium, respectively.
Tris(methylthio)methane and 34S ammonium sulfate were purchased from Sigma-Aldrich (Germany).