1% K2HPO4, 0.05% KCl, 0.05% MgSO4, 0.001% FeSO4) Crenigacestat chemical structure containing 200 μg/mL ammonium glufosinate and incubated at 27°C for 6-8 days. Transformants were confirmed by PCR amplification of bar gene. Post-transformation mitotic selleck stability was evaluated according

to the method in a previous report [46]. Quantification analysis of Ntl transcript Total RNA was isolated from mycelia using the Trizol reagent (Invitrogen, USA). The cDNA was synthesized from DNaseI-treated total RNA with an anchored oligo-dT primer following the manufacturer’s protocol (Promega, USA). Real-time PCR was performed using the SYBR-Green PCR Master Mix kit (Bio-Rad) in a Light Cycler (Bio-Rad). A standard curve was made to optimize the amplification efficiency with the primer pairs L1 (5′-GCACAAGAAGATACCGATGGC-3′) and L2 (5′-CGATCCACTGGGTTCTCATTTA-3′). Gdpdh encoding gly ceraldehyde-3-phosphate dehydrogenase was selected as an internal control, and the primers of 5′-AGATGGAGGAGTTGGTGTTG-3′ and 5′-GACTGCCCGCATTGAGAAG-3′ were used for it [47]. The cycling conditions were 95°C for 3 min followed by 45 cycles of 95°C for 10 sec, annealing at 59°C (Ntl) or 60°C (Gdpdh) for 10 sec. The relative expression level of the Ntl in M. acridum transformants compared to that in wild-type strain was determined with the comparative cycle threshold (CT) method [48]. Biological techniques were conducted in quadruplicate. Measurement of trehalose concentrations

and trehalase activity Trehalose levels in conidia were measured using a method modified from Foster et al. [28]. Conidia of both wild-type and M. acridum transformants were harvested from 14 day plates, Compound Library ic50 washed with distilled water, resuspended in 500 μL of water, boiled for 20 min, and disrupted by vortexing with glass beads (0.5 mm). Cell debris was removed by centrifugation at 13,000 g for 5 min and the supernatant was stored at 0°C prior to trehalose assay. A 50-μL

aliquot of the conidia lysis solution was added to 50 μL of 0.1 M sodium citrate buffer (pH 5.6). Duplicate samples were incubated with or without 10 μL porcine kidney acidic trehalase (Sigma, USA) overnight at 37°C. The reaction was stopped by boiling the sample for 10 min. Following centrifugation, the glucose concentration in the supernatant was assayed via a glucose assay kit (Bioscience, Quinapyramine China). To assay trehalase activity, 25 μL of the trehalase extraction solution were added to the trehalose solution containing 50 mM HEPS, and the mixture was incubated for 30 min at 37°C. The reaction was stopped by boiling the samples for 10 min, the samples were centrifuged, and the glucose in the supernatant was assayed using a commercial kit (Trinder, Sigma). Heat shock treatment Conidia were prepared as described above. For the wet-heat shock test, conidia were suspended in 1 mL sterilized water. The suspension was vigorously shaken and filtered through cotton cloth and diluted to a concentration of 1 × 107 conidia·mL-1.