Calcium involvement in dimorphism of Ophiostoma ulmi, the Dutch elm disease fungus, and characterization of calcium uptake by yeast cells and germ tubes

Abstract

Exogenous Ca2+, at concentrations up to 5 mM, induced partial germ tube formation in Ophiostoma (=Ceratocystis) ulmi in media normally supporting growth in the yeast-like phase. The calmodulin inhibitors calmidazolium (R24571) and trifluoperazine (TFP), and the Ca2+ionophore, A23187, suppressed germ tube formation in germ-tube-inducing medium without affecting yeast-like growth. R24571 was the most effective inhibitor, giving almost complete suppression at 3 μM. Addition of excess Ca2+ (up to 5 mM) did not reverse the inhibitory action of R24571 and only ∼ 10% of yeast-like cells formed germ tubes on addition of Ca2+ in the presence of 20 μM-TFP or 15 μM-A23187. Intracellular cAMP increased on incubation with R24571 and A23187, possibly as a result of inhibition of the cAMP phosphodiesterase. The exogenous supply of the calcium-binding agents methylhydroxybenzoate (MHB) and EGTA also suppressed germ tube formation under inducing conditions. These results confirm an involvement of Ca2+ in the yeast−mycelium transition of O. ulmi. Yeast-like cells and germ tubes of O. ulmi exhibited metabolism-dependent Ca2+ uptake which was reduced in the absence of glucose, or by the presence of KCN, the ATPase inhibitors N,N′-dicyclohexylcarbodiimide (DCCD) and diethylstilboestrol (DES), and the protonophoric uncoupler DNP, indicating dependence on the electrochemical proton gradient across the plasma membrane generated by the H+-ATPase. Germ tubes exhibited greater sensitivity to inhibitors of Ca2+ uptake than yeast-like cells, while Ca2+ uptake was competitively inhibited by Mg2+, Mn2+ and Zn2+. R24571 and A23187 inhibited Ca2+ uptake by germ tubes although TFP stimulated uptake in comparison to control cells. Ca2+ uptake by both cell types conformed to Michaelis−Menten kinetics at concentrations below ∼ 200 μM but deviated strongly above this concentration. Kinetic analysis of Ca2+ uptake by yeast-like cells and germ tubes, at Ca2+ concentrations below 100 μM, revealed that both cell types possessed Ca2+ transport systems of similar specificity, with K m values ranging between ∼ 15 and 25 μM, although germ tubes always exhibited greater Ca2+ uptake than yeast cells under similar experimental conditions, possibly a consequence of increased vacuolar compartmentation.