Cloning and characterization of metallothionein genes of ectomycorrhizal fungus hebeloma cylindrosporum

Abstract

Metallothioneins are a class of low molecular weight cysteine rich proteins that bind to large amounts of metal ions such as Zn2+, Cu2+, Cd2+ or Hg2+ (Hamer, 1986). MTs were identified from different filamentous fungi (Lanfranco et al., 2002). The occurrence of MTs and PCs or both, in ectomycorrhizal fungi still a matter of debate and the metal tolerance mechanisms are not well understood in mycorrhizal fungi. The molecular regulation of MTs and PCs has been investigated, to a limited extent in ectomycorrhizal fungi (Bellion et al., 2007). In the present investigation metallothionein genes of ectomycorrhizal fungus Hebeloma cylindrosporum were cloned and characterized. The expression of these genes was studied in the presence of different metals and their functions were assayed by yeast complementation. Axenic screening provides a simple and rapid process for determining fungal response to increasing metal doses, and identifying possible mechanisms of tolerance. In the present study, the tolerance levels of H. cylindrosporum to various concentrations of copper, cadmium, zinc, lead and nickel was assessed by growing pure mycelial cultures in liquid MMN media in vitro. The growth of H. cylindrosporum was adversely affected with increasing concentrations of metals. Among all metals tested, the H. cylindrosporum showed higher tolerance to Zn, Cu and Ni. The growth was inhibited 50% at 160µM of Cu and 170 µM of Ni respectively. There was no significant growth reduction observed in all concentrations of Zn tested. The H. cylindrosporum was very sensitive to Cd and Pb as their growth was completely inhibited at 14 µM of Cd and 50 µM Pb respectively. These results indicated that H. cylindrosporum showed large variation in metal tolerance to all metals tested and confers that the fungus is more tolerant to one or more types of metals not to other metals (Meharg, 2003).