Inhibition of Ornithine Decarboxylase and Growth of the Fungus Helminthosporium maydis

Abstract

a-DL-DifluoromethylomUithine (DFMO), a specific enzyme-activated inhibitor of ornithine decarboxylase, at 0.5 to 2.0 millimolar significantly inhibited mycelial growth and especially sporulation of Helminthosporium maydis in the dark; its inhibitory effect on sporulation was greatly increased under light conditions. Putrescine at 0.25 millimolar fully prevented the inhibitory effects of DFMO; the inhibition caused by the latter could not be prevented by cadaverine or CaC12. a-DL-Difluoromethylarginine, a, specific enzyme-activated inhibitor of arginine decarboxylase, at 0.1 to 2.0 millimolar had a weak inhibitory effect on the fungus. The effect was not dependent on the inhibitor concentration and there was no detectable arginine decarboxylase activity in the fungus.Our recent investigation on in vitro activities of ADC2 and ODC from higher plants (4,5) under the same conditions sporulation is three to four times as high as that found in cultures exposed to light (M. 0. Garraway, unpublished data). MATERIALS AND METHODSThe fungus was grown on a synthetic medium containing asparagine described previously (6). The sterilized agar culture media (20 ml per 100 x 20 mm sterile Petri dish) contained glucose, L-asparagine, KH2PO4, and MgSO4 at 0.5, 0.2, 0.3, and 0.15%. Sterilized solutions of Mn, Zn, Fe, and Cu salts at 1 ug/ 1 each were added at 0.2 ml per dish. Solutions of other compounds were filter sterilized prior to application. The pH of the culture media in experiment I was 5.0; in the remaining experiments it was adjusted to 6.0, which proved to be optimal for the fungal growth. Each Petri dish was inoculated at its center with a plug of mycelium 7 mm in diameter. Each experiment was carried out in four replicates. In experiments I and III the Petri dishes were placed in the dark at room temperature. In experiment II, 2 d after inoculation, the plates were transferred from darkness to diffused fluorescent light. Growth was determined by linear extension, and sporulation was expressed on dry weight basis (6).In experiment III the activities of ODC and ADC were determined after the enzymes were extracted with phosphate buffer (pH 7.6) (14) from mycelia exposed to the highest DFMO and DFMA concentrations. Repeated freezing and thawing, sonication, and maceration with glass beads was applied during extraction at 0 to 4°C. The enzymes were assayed as described previously (3,4)

The effects of polyamine biosynthesis inhibitors on mycelial growth, enzyme activity and polyamine levels in the oat-infecting fungus Pyrenophora avenae

“…This agrees with work using DFMO to inhibit fungal growth in vitro (Rajam & Galston, 1985 ;Birecka et al, 1986) and in viuo (Rajam et al, 1985;Walters, 1986). Treatment with DFMO strongly reduced ODC activities in P .…”

“…Increases in cell length and diameter were also reported in the presence of exogenous polyamines. As suggested by Birecka et al (1986) these differences may be accounted for by differing sensitivities between genera and perhaps also in the uptake and distribution of the inhibitor within the cell. The mechanisms underlying the differing fungal responses to inhibitors of polyamine biosynthesis are currently under investigation in this laboratory.…”

“…Studies using the inhibitor difluoromethylorni thine (DFMO), which irreversibly inhibits ODC (Metcalf et al, 1978), showed that DFMO was effective in controlling fungal growth in vitro. Reversal of inhibition was obtained by the addition of exogenous putrescine and spermidine (Birecka et al, 1986;Rajam & Galston, 1985).…”

First‐report of the inhibition of arbuscular mycorrhizal infection of Pisum sativum by specific and irreversible inhibition of polyamine biosynthesis or by gibberellic acid treatment

“…Higher plants and bacteria use both the ADC and ODC pathways for synthesising polyamines, while fungi possess only ODC for polyamine biosynthesis [3,4]. It should be possible, therefore, to control fungal polyamine biosynthesis by specifically inhibiting ODC without affecting plant growth and development [5][6][7][8].…”

Translational Modification of an 18 Kilodalton Polypeptide by Spermidine in Rice Cell Suspension Cultures

“…Recent evidence suggests a possible role for polyamines, especially Spd, in reproductive differentiation in various organisms (2,7,10,11,15 eluted with a protein fraction having a molecular mass in excess of 45,000 in oat and petunia protoplasts (14). Because there was a large difference in the relative molecular mass in the polypeptides from tobacco thin-layer culture and oat and petunia protoplasts, a need exists to establish unambiguously the size of proteins that are labeled by Spd, as was also suggested by Mizrahi et al (14).…”