cDNA was made with the mRNA as a template, and the relative this website expressions of the six putative trehalose synthesis genes, tpsA, tpsB, tpsC, tppA, tppB and tppC, were analyzed with real-time PCR. Figure 3 Expression of putative trehalose synthesis genes during outgrowth of A. niger conidia. The developmental stages are given on the x- axis: 0 h are dormant conidia; 3–72 h are swollen conidia, germlings or mycelia after so many hours of incubation in liquid AMM

media; and Plate is the entire sporulating culture grown on AMM plates for 5 days. Error bars show standard error of the mean based on four biological replicates each calculated as the average of three technical replicates. For all genes, the expressions are normalized against the expression

of actin. *Indicates that the expression at 0 h was statistically MK-1775 clinical trial significant to the following time-points within the same group except 3 h (one-way ANOVA, P < 0.05). **Indicates that the expressions at 0 h were statistically significant to all of the following time-points within the same group (one-way ANOVA, P < 0.05). The general expression pattern of the genes (Figure 3) was as follows: The expression was highest in still dormant conidia and had decreased by approximately 2-fold after 3 h incubation; after 6 h incubation there was a slight, but not significant, decrease; and, in 12 and 72 h mycelium the expression was very low. For tpsB, tppA and tppC, the expression was then up-regulated in sporulating colonies (5 days old), while it remained low for tpsC and tppB. One gene, tppA, deviated slightly from the described pattern: The decrease in expression after 3 h was not as profound as in the other genes, and a slight, but not significant, up-regulation could be seen in 72 h mycelium. Targeted gene deletions of six Aspergillus niger genes To characterize Bacterial neuraminidase the function of the six A. niger proteins, tpsA, tpsB, tpsC, tppA, tppB and tppC were all subjected to targeted gene deletions by replacing the gene with the A. oryzae pyrG resistance cassette. A double mutant, lacking the two adjacent genes tpsB

and tpsC was also constructed. All deletion mutants were confirmed with PCR using both internal and flanking primers (data not shown). With the exception of ΔtppA, all deletion mutants showed phenotypes similar to wild-type. When culturing the wild-types and mutants at temperatures ranging from 15°C to 37°C, no strain-dependent differences in growth rates or morphologies could be observed; at 10°C no growth was observed for any strain (data not shown). The tppA mutant showed a marked reduction in the number of conidia produced compared to the other Selleckchem RAD001 strains, giving the colonies growing on plate a whitish, and with age, light brownish appearance, compared to the black wild-type (Figure 4A,B). This phenotype was retained during aging, and under all growth conditions.