Statistical analyses were performed using the SAS version 9.2 (SAS Institute Inc., USA) applying the one-way ANOVA test. Ponatinib mechanism Means were compared using the Duncan’s multiple range test; when the P value was less than 0.05, the difference was regarded as statistically significant.2.5. Construction of Gene-Replacement Vector, pN1389PKACFor the construction of the CgPKAC replacement, the gene’s 5�� region (?643 to +52) and 3�� region (+930 to +1306) were amplified from pGEM-PKAC. Both fragments were subcloned into vector pN1389 carrying a hygromycin expression resistance gene, resulting in the pN1389PKAC replacement plasmid. The 5�� region was amplified by PCR with PKACpN5-F and PKACpN5-R primers (Table 1) containing terminal KpnI and BamHI sites, while the 3�� region was amplified with primers PKACpN3-F and PKACpN3-R (Table 1), containing terminal SdaI and SphI restriction sites, respectively.

Both fragments were cloned into pGEM-T Easy, resulting in pGEM-PKAC5�� and pGEM-PKAC3��. Subsequently, Plasmid pN1389 was digested with KpnI/BamHI and ligated with 695bp of the CgPKAC 5�� region fragment to produce plasmid pN1389PKAC5. Following that, pN1389PKAC5 was digested with SdaI/SphI and ligated with 376bp of the CgPKAC 3�� region fragment to produce the gene-replacement vector, pN1389-PKAC.2.6. Transformation-Mediated Gene ReplacementPreparation of spheroplasts and transformation of C. gloeosporioides were performed according to methods described by Rodriguez and Redman [17]. Transformants were selected on regeneration medium containing hygromycin B (300��gmL?1) (Sigma, USA).

Before transformation, pN1389-PKAC was linearized with the Kpn1 restriction endonuclease and precipitated with ethanol. Subsequently 20��g of DNA were transformed into C. gloeosporioides sphaeroplasts.2.7. Genomic DNA and RNA Blot AnalysesDNA digestion, agarose gel fractionation, labeling of probes, and hybridization were performed according to Cilengitide the manufacturer’s instructions and standard methods [18]. The 2.5kb of full length CgPKAC or 695bp of the 5��CgPKAC fragment were labeled with [��-32P] dCTP using the Ready To Go DNA Labeling kit (-dCTP) (Amersham, USA). Hybridization was carried out with hybridization buffer [1M Na2HPO4?2H2O, 1M NaH2PO4, 0.5M EDTA, 0.1% (w/v) SDS] at 65��C for 4h for prehybridization and hybridized overnight after the labeled probes were added. The membrane was washed at 65��C with 2X SSC for 10min followed by 2X SSC and 0.1% SDS, 1X SSC and 0.1% SDS, and finally 0.5X SSC and 0.1% SDS until the radioactivity signal was low. The washed blots were exposed to Fuji film for various times at ?80��C.2.8. Appressorium Induction on a Hydrophobic Hard SurfaceInduction of appressorium was tested on a glass slide coated with rubber wax.