Electron micrographs were acquired from uncoated frozen samples, or after sputter-coating with
check details gold three times during 30 s. Micrographs of uncoated samples were taken at an acceleration voltage of 2.5 kV, and consisted of 30 averaged fast scans (SCAN 2 mode). Coated samples were observed at 5 kV using F4 scans. Extraction of nucleic acids DNA was extracted as previously described . RNA from dormant conidia and conidia in early stages of germination (0 and 3 h) was extracted MAPK inhibitor according to Leeuwen and co-workers . RNA from germinating spores (6 and 12 h), mycelia and sporulating mycelia (plate) were extracted according to Plumridge and co-workers . As a final step in both protocols,
the RNA products were purified using a Qiagen RNeasy Mini kit (RNA clean up protocol). Two-hybrid assay The two-hybrid assay was performed using the BACTH System Kit (Euromedex). Full-length cDNA for all six genes were amplified using primers with Selleckchem Mocetinostat internal restriction sites (Table 2). After digestion of the PCR products, the inserts were ligated into linearized and dephosphorylated pKT25 and pUT18C
vectors and used to transform E. coli. All ligations in this work were performed with the ReadyToGo ligation kit (GE Healthcare) and were transformed into NEB 10-β Competent E. coli cells (New England Biolabs), unless otherwise stated. Correct insertions Farnesyltransferase were confirmed with vector specific primers (Table 2) followed by sequencing. Successful clones were co-transformed into electrocompetent BTH101 cells and selected on LA plates supplemented with ampicillin (100 μg/ml) and kanamycin (50 μg/ml). The protein-protein interactions were assayed according to the manufacturer’s protocol with the following modifications. One fresh colony of each interaction was transferred to 100 ml conical flasks with 5 ml LB supplemented with ampicillin 50 μg/ml, kanamycin 50 μg/ml and 0.5 mM IPTG, and incubated with shaking at 100 rpm at 20°C for 72 h. The extent of protein-protein interaction was measured with β-galactosidase assays as units/mg dry weight.