EVIDENCE FOR AN INOSITOL LIPID SIGNAL PATHWAY IN THE YEAST-MYCELIUM TRANSITION OF OPHIOSTOMA-ULMI, THE DUTCH ELM DISEASE FUNGUS

Results presented in this paper indicate the involvement of an inositol lipid signalling system, including protein kinase C, in the yeast-mycelium transition of Ophiostoma ulmi. The exogenous supply of inositol 1,4,5-trisphosphate (Ins1,4,5P3) induced germ tube formation in O. ulmi, maximum germ tube formation resulting at a concentration of 30-mu-m. The addition of Ins1,4,5P3 10 min after initiation of Ca2+ uptake by non-growing yeast cells of O. ulmi resulted in a slight increase in Ca2+ uptake. The protein kinase C-activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), also induced a significant germination response in O. ulmi and approximately 50% of the population exhibited germ tube formation after 12 h incubation in the presence of 16 nM-PMA. This indicated a possible role for protein kinase C, and therefore diacylglycerol, in the germination response. Ca2+ uptake by yeast extract-(1% w/v) induced germ tubes of O. ulmi was slower than that which occurred in PMA- or Ins1,4,5P3-induced germ tubes under similar conditions, PMA-induced germ tubes showing the highest levels of Ca2+ uptake. The anti-calmodulin drug R24571, at 3-mu-M, caused almost complete suppression of a yeast extract-induced yeast-mycelium (Y-M) transition which confirmed the involvement of calmodulin in germ tube formation. The addition of 16 nM-PMA in conjunction with yeast extract had no effect on germ tube formation yet, when added with R24571, the inhibitory effect of R24571 was reversed and an almost complete Y-M transition resulted after 16 h incubation. This provided further evidence that activation of protein kinase C was required for germ tube formation in O. ulmi. Ca2+ uptake by yeast cells or during the growth of yeast extract- and PMA-induced germ tubes of O. ulmi increased markedly over the first 12 h of growth with highest Ca2+ uptake occurring in yeast extract-induced germ tubes. Ca2+ uptake by 12 h yeast cells of O. ulmi was inhibited after preincubation for 10 min with PMA suggesting either inhibition of Ca2+ influx or stimulation of Ca2+ efflux. Use of Ca2+-loaded cells showed that efflux of Ca2+ from Ca2+-loaded yeast cells of O. ulmi was similar in the absence or presence of 70 mM glucose but increased rapidly on addition of PMA to the incubation medium.