Hygromycin b

Human immortalized fibroblasts BJ (ATCC, Germany) were cultured in media consisted of DMEM (Gibco) and Medium 199 (Sigma-Aldrich) in 4:1 proportion containing 10% FBS (AG Biochrom), 10 mM glucose, 3 mM glutamine, 1 mM pyruvate, 0.085 mg/mL hygromycin B (Roche), 0.4 mg/mL puromycin (Sigma-Aldrich) and 1% antibiotic. In our experiments the passage number never exceeded 10.

The full-length coding sequences of AtMinD1 were amplified using the primer pair W-001/W-002. The PCR product is fused into the NcoI/BstEII site of pCAMBIA1302 through digestion. pCAMBIA1302-AtMinD1 was employed for Agrobacterium-mediated Arabidopsis transformation with the floral dip method (Clough and Bent, 1998 (link); Itoh et al., 2001 (link)). In total, 32 transformed (T1) seedlings were selected on MS plates containing hygromycin-B (25 mg/L; Roche, Germany), and T2 to T4 progenies were used for microscopic characterization. Stable T4 seedlings, grown on antibiotic-free MS plates, were subjected to quantitative RT-PCR analysis using the primer pair HY-053/HY-054.

Mtb H37Rv (ATCC 27294) and M. bovis BCG (ATCC 35734) strains were maintained in complete Middlebrook 7H9 medium (BD Difco) supplemented with 0.05% (vol/vol) Tween 80, 0.5% (vol/vol) glycerol, 0.5% albumin, 0.2% glucose, 0.085% sodium chloride, and 0.0003% catalase at 310 K with agitation at 80 rpm. POA-resistant strain Mtb H37Rv POA^R18 (clpC1: A625G/Lys209Glu) was isolated and described previously⁷ . The Mtb H37Rv ΔprcAB strain (hygromycin-resistant) and its complemented mutant (hygromycin-resistant and Kanamycin-resistant) were previously generated and described^{18 (link)}. Hygromycin B (Roche) and Kanamycin (Sigma-Aldrich) were used for selection when required at 50 and 25 µg/mL, respectively. PZA, POA, and Isoniazid were purchased from Sigma-Aldrich. Bortezomib was purchased from Chembridge. Antibiotics were dissolved in 90% DMSO and sterilized using 0.2 μm PTFE membrane filters (Acrodisc PALL).

C. lunata CX-3 provided by Prof. Chen (Shanghai Jiaotong University, Shanghai, China) was used as the wild-type strain and was maintained on potato dextrose agar (PDA) at 28 °C. The selective medium was supplemented with either 250 μg/ml of hygromycin B (50 mg/ml, Roche Applied Science) or 200 μg/ml of Neomycin (Sigma-Aldrich Co. LLC) according to the selection marker gene.

To confirm the interactions of Ebg1 and EF1α in vivo, the genomic sequence of EBG1 was cloned into pGTN containing a GFP tag under its native promoter, while the genomic sequence of EF1α was cloned into pTH3Flag under its native promoter (Supplementary Table 3). The resulting constructs pGTN-EBG1 and pTH3Flag-EF1α were co-transformed into protoplasts of the wild-type strain P131. Transformants were screened on CM plates containing both neomycin (400 μg/ml, Ameresco) and hygromycin B (250 μg/ml, Roche). Total proteins from culture filtrates (CM) of the transformant expressing both Ebg1-GFP and EF1α-3Flag were incubated, respectively, with anti-GFP affinity resins (Chromotek, gta-20) and anti-Flag M2 affinity resins (Sigma-Aldrich, F2426). Proteins bound to resins were eluted after a series of washing steps following the manufacturer’s instruction. The total proteins and elution from resins were detected by western blotting.
For western blotting detecting the presence of proteins, the following antibodes were used, anti-Actin (ABclonal, AC009, 1:2500), anti-Flag (Sigma, A8592, 1:5000), anti-HA (Sigma, H3663, 1:5000), anti-His (Abmart, 10E2, 1:5000), anti-p44/42 (Cell signaling, 9101, 1:2500), anti-GFP (Abclonal, AE012, 1:5000), anti-GST (EASYBIO, BE7012, 1:5000).

The wild type M. oryzae strain Guy11 and its derivative strains were grown on complete medium (CM) plates [20 (link)] at 28°C. Conidia were harvested from 9-day-old cultures grown on CM plates. Genomic DNA and total RNA were extracted from 3-day-old cultures grown in liquid CM. Fungal transformants were screened in CM plates supplemented with 200 μg ml⁻¹ of hygromycin B (Roche Applied Science, Mannheim, Germany) or 250 μg ml⁻¹ of glufosinate ammonium (Sigma-Aldrich, St Louis, MO, USA), depending on the selection marker. Evaluations of conidiation and vegetative growth were performed as previously described [21 (link)]. And growth rate of M. oryzae was determined by measuring the colony diameter of 6-day-old cultures on CM plates supplemented with chemicals.