Identification and characterization of eight metallothionein genes involved in heavy metal tolerance from the ectomycorrhizal fungus Laccaria bicolor

Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification. The increasing numbers of available genomic sequences of ectomycorrhizal (ECM) fungi enable deeper insights into the characteristics of MT genes in these fungi that form the most important symbiosis with the host trees in forest ecosystems. The aim of this study was to establish a comprehensive, genome-wide inventory of MT genes from the ECM fungus Laccaria bicolor. Eight MT genes in L. bicolor were cloned, and the expression patterns of their transcripts at various developmental stages based on expressed sequence tag (EST) counts were analyzed. The expression levels of four MTs were significantly increased during symbiosis stages. Quantitative real-time PCR (qRT-PCR) analysis revealed that transcripts of LbMT1 were dominant in free-living mycelia and strongly induced by excessive copper (Cu), cadmium (Cd), and hydrogen peroxide (H 2 O 2 ). To determine whether these eight MTs functioned as metal chelators, we expressed them in the Cu- and Cd-sensitive yeast mutants, cup1∆ and yap1∆ , respectively. All LbMT proteins provided similar levels of Cu(II) or Cd(II) tolerance, but did not affect by H 2 O 2 . Our findings provide novel data on the evolution and diversification of fungal MT gene duplicates, a valuable resource for understanding the vast array of biological processes in which these proteins are involved.