Calcium-regulated appressorium formation of the entomopathogenic fungusZoophthora radicans

Summary The fungusZoophthora radicans (Zygomycetes: Entomophthorales) requires external Ca²⁺ for appressorium formation but not for conidial germination. The number of appressoria formed depends on the Ca²⁺ concentration of the medium. At low Ca²⁺] (100 pM) nuclear division and germ tube growth are significantly reduced compared to higher Ca²⁺ concentrations (10 and 1,000 M). By contrast, neither external K⁺ nor external Cl^– is needed for germination or appressorium formation. Treatment of conidia with a Ca²⁺-antagonist, Nd³⁺, and a Ca²⁺-channel blocker, nifedipine, inhibits appressorium formation, showing that a Ca²⁺ influx is required for appressorium formation. Furthermore, the partial yet saturating inhibition by nifedipine and complete inhibition by Nd³⁺ indicates that at least two kinds of Ca²⁺ channels are involved in appressorium formation. A contribution of intracellular Ca²⁺ to the signal transduction chain for the formation of appressoria is demonstrated by the inhibitory effect of the intracellular Ca²⁺ antagonist TMB-8. The calmodulin antagonists R24571, TFP, W-7, and W-5 inhibit appressorium formation at concentrations which have no effect on germination. The data presented in this paper are consistent with the hypothesis that a Ca²⁺/calmodulin system is involved in regulating appressorium formation. However, since the direct effects of the drugs were not specifically tested on their proposed binding sites, we leave room for alternative hypotheses that have yet to be formulated.Abbreviations A-9-C 9-anthracenecarboxylic acid - DAPI 4,6 diamino-2-phenylindole - EGTA ethylene glycol bis(-aminoethylether)-N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - H-7 N-(2-methylamino)ethyl-5-isoquino-linesulphonamide dihydrochloride - IC₅₀ concentration of inhibitor that causes 50% inhibiton - R24571 (calmidazolium) 1-bis-(4-chlorophenyl)methyl]-3-2,4-dichloro--(2,4-dichlorobenzyloxy)phenethyl]-imidazolium chloride - TEA tetraethylammonium - TFP (trifluoperazine) 10-3-(4-methylpiperazine-1-yl)-propyl]-2-trifluomethylphenothiazine - TMB-8 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride - W-5 N-(6-aminohexyl)-1-naphthalene-sulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide