(A) A total of 2 × 103 conidia were point inoculated on agar plates (CM for GR5, RhoAG14V, RhoAE40I and ΔmpkA, repressive MM containing 1% glucose according to [26] for R135 and alcA-PkcA) containing the appropriate supplements and 0, 0.2 and 1 μg/ml AFPNN5353 for GR5, RhoAG14V, RhoAE40I, R135 and alcA-PkcA. The ΔmpkA mutant and its reference strain GR5 were exposed to 0, 0.5 and 1 μg/ml AFPNN5353. The plates were incubated at 37°C for 48 h. (B) 1 × 104 conidia/ml of the ΔmpkA mutant and GR5 were treated with 0.05 μg/ml AFPNN5353 or without protein (controls) in a total Tamoxifen manufacturer volume of 200 μl of appropriately supplemented CM in

96-well plates. In addition, mutants defective in PkcA and MpkA activity were tested for their AFPNN5353 susceptibility. As pkcA is an essential gene in A. nidulans, a conditional alcA-PKC mutant strain was used, where the pkcA gene was put under the control of the alcA promoter, which is repressed by glucose but derepressed by glycerol [26]. Both the conditional alcA-PKC mutant (cultivated under repressive conditions) and a ΔmpkA mutant were hypersensitive to AFPNN5353 compared to their recipient strains R153 and GR5, respectively, indicating that the activity of PkcA and MpkA confers a certain resistance to AFPNN5353 (Figure 2A). The hypersensitive phenotype of the ΔmpkA mutant was also confirmed by liquid growth inhibitory assays. In unchallenged

liquid condition, the GR5 and the ΔmpkA mutant showed a comparable proliferation rate (Figure 2B).

In the presence of 0.05 μg/ml AFPNN5353, however, the mpkA deletion strain did not germinate Alvelestat molecular weight whereas the GR5 strain still exhibited 11% growth. Note that growth inhibition in liquid conditions requires less antifungal protein to monitor its toxicity than on solid media probably due to less diffusion in the latter case (data not shown). From these data we conclude that AFPNN5353 interferes with the cell wall homeostasis of A. nidulans and that this interaction is mediated by PkcA/MpkA signalling, although independently from RhoA. AFPNN5353 disrupts calcium homeostasis in A. niger Supplements other than osmotic stabilizers can also antagonize the activity of antifungal proteins from plants and ascomycetes. Baricitinib For example, the addition of cations such as Ca2+ ions to the growth medium reversed the antifungal activity of the P. chrysogenum PAF [17], the A. giganteus AFP [15, 21] and of plant defensins [29, 30] which are usually positively charged due to their high pI. A cation-sensitive antifungal mode of action can for example be associated with the perturbation of the intracellular Ca2+ homeostasis by antifungal peptides [17, 18] but might also result from the interference of cations with antifungal-target interaction(s). Therefore, we tested to which extend these effects also account for the antifungal activity of AFPNN5353. To this end, we selected A.