A variety of morphological abnormalities were observed in the MaAC RNAi mutants. On PDA, the growth of the MaAC RNAi mutants was reduced, mycelium formation was delayed, and the colonies of RNAi mutants were Ro 61-8048 cell line smaller compared to the wild type. On Czapek-dox medium, the conidiation of the MaAC RNAi mutants was also delayed, and the colonies of RNAi mutants were lighter in comparison this website to the wild type. The AC-RNAi-3 mutant had the most significant difference compared to the wild type,

and was used as the MaAC RNAi mutant in the following experiments. Figure 3 Effect of  MaAC  on vegetative growth in the wild type and AC-RNAi mutants. A. The colonies were cultured on PDA and Czapek-dox medium for 10 d. Scale bar: 0.5 cm. B. The OD490 after a 3-h incubation of the wild type and AC-RNAi mutant cultured for 72 h mixed with CellTiter 96® AQueous One Solution Reagent in PD liquid culture. Error bars denote the standard deviations from three trials. Vegetative growth in vitro was further quantified by assaying the living cells in PD liquid culture by CellTiter 96® AQueous One Solution Assay (Figure 3B). In contrast to

the wild type, the growth rate of the AC-RNAi-1 mutant was similar to the wild type, while the other four RNA mutants grew conspicuously slowly (p <0.01). These results indicated that MaAC affects growth in vitro. The correlation coefficient of the

relative expression rate and the growth rate was 0.94, which was highly significant (p <0.01). These result showed that the growth rate is related to the relative expression Belnacasan rate of MaAC. MaAC regulates intracellular cAMP levels in M. acridum As shown in this study, the fungal growth of the MaAC RNAi mutant of M. acridum was significantly slower in vitro than that of the wild type. In order to assess whether the growth defect of the RNAi mutant was due to reduced levels of cAMP, we quantified and compared the steady-state levels either of cAMP in PD liquid culture. The cAMP level was significantly reduced in the AC-RNAi-3 mutant compared to the wild type (Figure 4A) and the cAMP concentration of the MaAC RNAi mutant (259.4 fMol/mg) was approximately two-fold less than that of the wild type (486.8 fMol/mg) after being cultured for 30 h (p <0.01). This demonstrated that MaAC was involved in cAMP production during the vegetative growth of M. acridum. This was further confirmed by the exogenous addition of cAMP (8-Br-cAMP) to the RNAi mutant. As shown in Figure 5, the RNAi mutant grown in the presence of 8-Br-cAMP showed a great increase in aerial hyphal growth. Thus, exogenous cAMP could restore the growth of the RNAi mutant, which suggested that MaAC was involved in cAMP synthesis. Figure 4 cAMP levels in the AC-RNAi mutant and wild type strains.