heterostrophus genomic DNA as template. Reactions with pairs 3 and 4 were carried out using pATBS-NEO (M. Ronen, PhD thesis, Technion, 2011) plasmid DNA as template. Round-II used, to construct the 5′ side of the final sequence, the products of pairs 1, 3 as template and FP1, NLC37 as primers; for the second half, the products of pairs 2 Depsipeptide cost and 4 as template and NLC38, RP2 as primers. The two final products were integrated into the Δskn7 genome by double-crossover recombination, resulting in reconstruction of the complete
neomycin resistance cassette replacing the entire predicted coding region of ChAP1. Fungal protoplasts were prepared and transformants selected for neomycin and hygromycin resistances as described (Turgeon et al., 2010; Turgeon et al., 1987; Wirsel et al., 1996). To assay gene expression, cultures were grown in liquid CMX with shaking NVP-BEZ235 (200 r.p.m.) for 4 days at 22 °C, the mycelium centrifuged and transferred to fresh CMX with 20 mM final concentration of hydrogen peroxide and incubated at 22 °C for 30 min. RNA isolation was done as described in (Shanmugam
et al., 2010). For cDNA synthesis, 2 μg of RNA was used for reverse transcription with random primers following the protocol supplied with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Abundance of transcripts was measured by quantitative real-time PCR in a 7300 cycler (Applied Biosystems), with 15 μL reaction volumes, using Quanta Biosciences SYBR mix with two technical replicates for each PCR reaction. Data shown are means of three biological replicate experiments. The C. heterostrophus actin gene was used as ‘housekeeping’ gene to normalize the amount of cDNA. The primers used for real-time PCR are shown in Table 2. Calculation of CT values was done using Applied Biosystems software dataassist. Solid CMX was amended with 20 mM hydrogen peroxide, 0.4 M potassium acetylcholine chloride, 0.75 M sorbitol, 30 μM menadione or 25 mM calcofluor white stain (CWS). Control was solid CMX without additives. All plates were incubated
under 16 h light–8 h dark at 22 °C for 6 days, and colonies were photographed. Sorbitol, calcofluor white stain, menadione, and MES hydrate were purchased from Sigma-Aldrich. Hydrogen peroxide was purchased from Carlo Erba. Potassium chloride was purchased from MERCK. Murashige and Skoog medium was purchased from Duchefa Biochemie. Maize plants (Royalty, local hybrid, purchased from Ben Shachar, Tel Aviv) were grown in hydroponic culture for 12 days in a medium containing 2.15 g L−1 of Murashige & Skoog medium (0.5 MS), 0.25 mM MES, adjusted to pH 5.7 with KOH. Plants – with their roots – were attached to a tray and kept moist. The second leaf was inoculated with 7-μL droplets of 0.02% Tween 20 in ddW containing about 500 C. heterostrophus spores. Trays were closed in plastic bags to keep the plants moist. Lesions were measured after 2 days.