2 μg/ml for A. nidulans, 0.5 μg/ml for N. crassa and 1 μg/ml for A. niger. Two strains PD0332991 concentration were unaffected at the protein concentrations tested:

M. circenelloides and M. genevensis were insensitive against AFPNN5353 when concentrations up to 500 μg/ml were used. Table 1 Minimal BAY 57-1293 order inhibitory concentrations (MIC; μg/ml) of AFPNN5353 against different filamentous fungi. organism MIC (μg/ml) Aspergillus flavus ATCC9643 50 Aspergillus fumigatus ATCC 46645 50 Aspergillus giganteus AG090701 50 Aspergillus nidulans FGSC4 0.2 Aspergillus niger CBS 120.49 1 Aspergillus terreus 304 5 Botrytis cinerea BC 080801 10 Fusarium oxysporum FO 240901 5 Fusarium sambucinum FS210901 5 Gliocladium roseum GR 210901 100 Mucor circinelloides MC080801 insensitivea Mucor genevensis MG 080801 insensitivea Penicillium chrysogenum ATCC10002 10 Trichoderma koningii TC 060901 20 Neuropsora crassa FGSC 2489 0.5 aup to 500 μg/ml AFPNN5353 was tested 1

× 104 conidia/ml were incubated in 200 μl CM medium in the presence of various concentrations of AFPNN5353 at 30°C for 24 h. Growth was determined by measuring the OD620 nm. Z-IETD-FMK mw AFPNN5353 interferes with the cell wall integrity of A. nidulans It is known that antifungal compounds such as congo red, caffeine, CFW or caspofungin interfere with cell wall biosynthesis and weaken the cell wall in fungi (reviewed by [24]). The remodeling of the cell wall by these antifungal compounds is mediated by the activation of the CWIP. In fungi, extracellular signals are transmitted via the membrane bound small unless GTPase RhoA to the central regulators Pkc and Mpk, which are regulated by phosphorylation/dephosphorylation. The signal transduction cascade eventually enforces transcription of cell wall synthesis genes, partly via the transcription factor RlmA [16, 25]. Respective loss-of-function or conditional mutants show hypersensitive phenotypes in the presence of cell wall perturbing agents [[9, 24–26]]. Similar to substances that weaken the cell wall, the A. giganteus antifungal protein AFP modulates the cell wall composition by inhibiting chitin

synthesis in sensitive fungi (e.g. A. niger, A. oryzae) and inducing the expression of agsA most likely by the activation of the CWIP [10]. To study the involvement of the CWIP in AFPNN5353 toxicity, we first tested whether the osmotic stabilizer sorbitol counteracts the toxicity of AFPNN5353. In the absence of AFPNN5353 A. nidulans proliferated less well in the presence of 1 M sorbitol and reached only 30% growth compared to the growth in standard medium (100%). Nevertheless, the addition of 1 M sorbitol to the growth medium strongly reduced the activity of AFPNN5353 on A. nidulans wild type. The osmotic stabilizer ameliorated growth in the presence of 0.05 μg/ml AFPNN5353 by 80% compared to a 10% growth rate in the absence of sorbitol (Table 2). This was even more accentuated when 0.1 and 0.