Developmental stages included M (mycelia harvested three days post inoculation), CM (mycelia harvested 10 days post inoculation), AH, and GC (24 h post inoculation of conidia in liquid SMS). For interactions, C. rosea was confronted with B. cinerea (Cr-Bc) or F. graminearum (Cr-Fg) on agar plates and the growing front (7-10 mm) of C. rosea was harvested before contact (5-7 mm apart), at contact, and post 24 h
contact. C. rosea confronted with itself (Cr-Cr) was used as control treatment. For interaction with barley roots, surface sterile seeds MI-503 were germinated on sterile filter paper placed on water agar (5 seeds per replicate). C. rosea conidia (1e + 07) were inoculated five days post germination and were allowed to interact for five days before harvesting of roots along with fungal Selleckchem PHA-848125 mycelium. Harvested samples were immediately frozen in liquid nitrogen and stored at -80°C. RNA extraction from all samples was done using the Qiagen RNeasy kit following the manufacturer’s protocol (Qiagen, Hilden, Germany). RNA was treated with RNase-free DNaseI (Fermentas, St. Leon-Rot, Germany) and concentrations were determined
spectrophotometrically CHIR-99021 supplier using NanoDrop (Thermo Scientific, Wilmington, DE). One or two microgram of total RNA was reverse transcribed in a total volume of 20 μl using the Maxima first stand cDNA synthesis kit (Fermentas, St. Leon-Rot, Germany). Transcript levels were quantified by qPCR using the SYBR Green PCR Master Mix (Fermentas,
St. Leon-Rot, Germany) in an iQ5 qPCR System (Bio-Rad, Hercules, Loperamide CA) as described previously [50]. Melt curve analysis was performed after the qPCR reactions, to confirm that the signal was the result from a single product amplification. Relative expression levels for target genes in relation to tubulin expression [51] were calculated from the Ct values and the primer amplification efficiencies by using the formula described by Pfaffl [52]. Gene expression analysis was carried out in 3 biological replicates, each based on 2 technical replicates. Primer sequences used for gene expression analysis are given in Additional file 1: Table S2. Construction of disruption and complementation vectors Genomic DNA was isolated using a hexadecyltrimethylammonium bromide (CTAB)-based method [53]. Phusion DNA polymerase (Finnzymes, Vantaa, Finland) was used for PCR amplification of a 1 kb 5′-flank and 3′-flank region of the Hyd1, Hyd2 and Hyd3 genes from genomic DNA of C. rosea using primer pairs Hyd1 ko-1 F/1R and Hyd1 ko-2 F/2R; Hyd2 ko-1 F/1R and Hyd2 ko-2 F/2R; and Hyd3 ko-1 F/1R and Hyd3 ko-2 F/2R, respectively (Additional file 1: Table S2). The hygromycin resistance gene (hygB) cassette was amplified from the pCT74 vector [54] using the P3/P4 primer pair (Additional file 1: Table S2).