Characterization Of Eight Metallothionein (MT) Genes In The Ectomycorrhizal Fungus Laccaria Bicolor And Identification Of A Novel Fungal MT Family

Mycorrhizae is a mutualistic symbiont formed by plant roots and some fungi in the soil. Ectomycorrhizas (ECMs), key members of the mycorrhizal family, are widely distributed in nature. They mainly belong to ascomycotina and basidiomycotina, in which basidiomycotina predominates. Metallothioneins (MTs) are low-molecular-weight and Cys-rich metal binding proteins, which are widely distributed in the genome of prokaryotes, fungi, plants and animals. The main functions of MTs are toxic metal detoxification, reactive oxygen species scavenging, and metallochaperone functions in the homeostasis of both copper and zinc. The currently available fungi MT genes mainly origin from ascomycota, with increasing numbers of fungi genomes have been released, MTs from various taxonomic groups can be retrieved exhaustively.In this research, we identified and cloned 8 MT genes from the ectomycorrhizal basidiomycete fungus Laccaria bicolor S238N. The genome analysis showed that except LbMTl, the other 7 LbMTs, which sharing high similarity, possess a large number of transposable elements within 60 Kb upstream and downstream range. We assume these MTs are formed by recent gene duplications. Through the complementary expression experiments in Cu-, Cd-and H2O2-sensitive yeast mutants, we found that all the 8 LbMTs have stronger chelating ability regarding to Cu and Cd, but they don’t have anaplerosis ability under the H2O2 stress, which suggests they couldn’t participate in the cell H2O2 clearance directly. In the transcription level, the expression level of LbMT2a, LbMT2b, LbMT3c and LbMT4 are significantly higher than the other LbMTs during the L. bicolor and plant roots symbiosis. Further bioinformatics analysis of the 1500 bp regions upstream of these 8 genes suggested that the promoter regions of LbMT2a, LbMT2b, LbMT3c and LbMT4 possess multiple transcriptional factor binding sites which are related to nutrients responding. In the meantime, we studied the expression profile of the single copy gene LbMT1 under Cu, Cd, Zn and H2O2 stress. qPCR results showed that the transcription products of LbMT1 have a relatively high abundance in free-living mycelium, and compared to other kinds of stress, its expression level only increased remarkably under high concentrate Cu stress condition.On the other hand, we used known basidiomycotina MT genes as probes to perform large-scale alignments, splicing, searching for ORFs and eliminating redundancy using public available fungi genome and transcriptome database of NCBI and JGI. After that, we found a novel MT subfamily widely distributed in basidiomycetes genome with three common features:1) possessing 3 to 4 exons and the first exon is usually very short (3-9 bases); 2) able to chelating heavy metals including Cu and Cd; 3) having a conserved 7-Cys residues domain in the C-terminal. This is the first time fungi MT genes are found widely existing in phyla other than ascomycotina, and the expression level of multiple MT genes of this kind were reported to change remarkably during the symbiosis between fungi and plants. The identification of this new fungi MTs family provides important clues for studying the specific function and regulation mechanisms of this kind of peptides in plant-fungi interactions.