The expression of tppB was selleck chemicals examined in mycelium from wild-type, ΔtppB and tppB+. In the deletion mutant, no expression was detected, whereas in the complemented strain, the levels were in the same range as in the wild-type
(Figure 8B). From these experiments we concluded that the deletion of tppB causes the lowered trehalose levels in ΔtppB. However, since the plasmid carrying the wild-type version of the gene was lost in most conidia, the tppB+ strain was not included in the following experiments. Figure 8 Trehalose content of mycelium (A) and relative expression EPZ-6438 research buy of tppB (B). Error bars show standard error of the mean, based on three biological replicates, and for qPCR each biological replicate was calculated as the average of three technical replicates. To evaluate the importance of trehalose as a stress protectant, the trehalose contents of the ΔtppB mutant and the control strains were analyzed in early stages of germination, and were subjected to lethal and sub-lethal heat and oxidative stress as well as sub-lethal salt and acid stress. The trehalose levels in ΔtppB followed the same pattern of breakdown and re-synthesis as in the control strains, but they were consistently
lower in accordance with the lower initial value (Figure 9). Dormant conidia of ΔtppB were significantly less tolerant to heat stress compared to the control strains; After 60 min of heat stress, the survival of ΔtppB was 35% compared to 78% in wild-type. After an additional 60 min, Plasmin the survival of Tucidinostat order ΔtppB further decreased to 2%
compared to 38% in wild-type. (Figure 10). These experiments were repeated with the new independent deletion mutant, ΔtppB2, and the results were identical to those for ΔtppB (data not shown). For the other stressors tested, benzoic acid, NaCl and H2O2, as well as long-term viability where conidia were stored in water at 4°C for a total of 8 weeks, no significant differences between the mutant and the control strains could be detected (data not shown). Figure 9 Concentration of trehalose during outgrowth of wild-type, pyrG + and ΔtppB conidia. Note the scale break between 12 and 72 h and that pyrG + observations are horizontally offset to avoid visual overlap. The error bars represent the standard error of the mean. The level of trehalose in ΔtppB was significantly different compared to wild-type for all time points except 3 h (two-way ANOVA, P < 0.0001 at 0, 6 and 12 h, and P < 0.01 at 72 h). Figure 10 Viabilities of dormant A. niger conidia after subjection to heat stress. Conidia were held at 55°C for 20, 60, 90 and 120 min. For all strains, the numbers of counted colonies were normalized to 25 at time = 0 min to avoid differences in numbers of assayed spores. Note that pyrG + observations are horizontally offset to avoid visual overlap. There were no significant differences between the control strains (N402 and pyrG+).