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). Two partial cDNAs encoding metallothionein (MT) like polypeptides designated HcMT1 and HcMT2 were identified from an EST library of the ectomycorrhizal fungus H. cylindrosporum (Lambiliotte et al., 2004) and primers designed (HcMT1F and HcMT1R and HcMT2F and HcMT2R) for the amplification of metallothionein gene. Genomic DNA was isolated from H. cylindrosporum. Total RNA was isolated and cDNA prepared by RT-PCR method. Both cDNA and genomic DNA were used for PCR to amplify metallothionein genes. The results showed that HcMT1F and HcMT1R primers amplified the 490 bp long fragment of genomic DNA and 295 bp of cDNA. In the case of HcMT2F and HcMT2R primers, 295 bp long fragment of genomic DNA and 210 bp of cDNA were obtained. Corresponding full length of HcMT1 and HcMT2 were obtained by RACE PCR according to manufactures instruction (5’/3’ RACE kit, Roche, USA). The PCR products amplified from genomic DNA and cDNA were cloned and transformed into DH5α E. coli cells by the heat shock method. The positive clones were selected and the plasmid DNA was isolated. Plasmid containing inserts were confirmed by PCR with insert specific primers and sequenced. Sequence analysis was performed with BLAST program (Altschul et al., 1997) using the nucleic acid and predicted amino acid sequences deposited in multiple data bases. The HcMT1 and HcMT2 contained 4 exons and 3 introns and all the 3 introns contained conserved intron junctions GT-AG. The ORF of HcMT1 and HcMT2 contained 177 bp (59 amino acids) and 174 bp (57 amino acids) respectively. HcMT1 contains 13 Cys residues and two C-x-C motifs at N-terminal part and three at their C-terminal portion, which is characterization of MTs. The HcMT2 sequence contains 14 cystein and six C-x-C motifs are equally distributed at both ends. HcMT1 and HcMT2 were 31% identical to each other and the similarity was 40%. The alignment of HcMT1 and HcMT2 with other known fungal MTs showed that fungal MTs bear the C-x-C-x(2,3)-C signature at the N-terminal part together with a conserved Cys residue at the C-terminal end. This consensus sequence is far more restricted than the C-G-C-S-x(4)-C-x-C-x(3,4)-C-S-x-C consensus proposed as a signature of fungal MTs by Binz and Kägi (1999) (http://www.expasy.ch/cgi-bin/lists?metallo.txt). The alignment of sequence shows that this latter consensus sequence resembles the signature of basidiomycete MTs, which is C-x(3, 4)-C-x-C-x(3)-C-x-C at the N-terminal end together with C-x-C at the C-terminal end. The genomic clones of HcMT1 and HcMT2 and the adjacent DNA sequence, 1200 kb upstream from HcMT1 and HcMT2 start codon of H. cylindrosporum was cloned using the Universal Genome Walker kit (Clonetech Laboratories, Inc., Germany). In order to explain whether the differential expression of HcMT1 and HcMT2 is due to differential regulation, we performed computational analysis of their respective upstream regions of the promoters. Both HcMT1 and HcMT2 promoters contained the standard stress response elements implicated in metal response such as metal response element (MRE), general stress response (GATA), response to phosphate starvation (PHO), response to nitrogen utilization (NIT), and heat shock (HSF). HcMT1 contained STRE (multiple stress response element), and GCN, which were not present in HcMT2 promoter region. The HcMT2 promoter contained several additional regulatory elements such as GCR, DRE, MCM, RPU and CAT. The difference and location of potential regulatory elements in HcMT1 and HcMT2 promoter regions might be the reason for different regulation pattern.

To study the metallothionein induction by different heavy metals, the fungus was first grown in Melin’s liquid medium for two weeks. Then the mycelium was transformed to fresh medium containing different concentrations of heavy metals. For dose responsive studies, the fungus was grown in different concentrations of Cu (0, 80, 160, 240 and 320 µM), Cd (0, 7, 14, 21 and 28 µM), Zn (0, 0.5, 1 and 1.5 mM), Ni (0, 85, 170, 250 and 350 µM) and Pb (0, 25, 50, 75 and 100 µM) for 24 hours. For induction kinetics studies, the fungus was grown in Melin’s medium with 320 µM of Cu and 21 µM of Cd concentration for different time intervals 0, 12, 24, 36, 48, 60 and 72 hours.

Competitive RT- PCR was used to quantify the mRNA accumulation of HcMT1 and HcMT2. The competitor sequence was plasmid cloned genomic DNA fragment. In a PCR reaction containing a RT-cDNA sample and a known amount of plasmid cloned competitor, primers HcMT1F and HcMT1R amplify simultaneously the 295bp long cDNA fragment and the 490 bp long competitor fragment. In the case of HcMT2, 210 bp long cDNA fragment and the 295 bp long competitor fragment was obtained with HcMT2F and HcMT2R primers. Standard curves were constructed by co-amplifying different known amounts of target DNA with a constant amount of competitor. A standard curve was obtained by plotting the log values of the amplification ratios of target DNA/competitor against the log values of the target DNA (pg of target DNA) added to PCR mix before amplification.

The kinetics of HcMT1 and HcMT2 transcript accumulation results showed that transcript accumulation was observed maximum at 24 hours and decreased thereafter. The highest mRNA accumulation was recorded with HcMT1 which was induced by ca. 350 fold at 24 hour treatment. HcMT2 was slightly less sensitive to Cu induction; at 24 hours, transcript level was ca. 100 times higher than control. HcMT2 was also induced by Cd. Transcript accumulation was maximum at 48 hour treatment. It was ca. 40 times as high as control (zero time). By contrast, HcMT1 was not induced by Cd.

Dose responsive studies showed that the induction of HcMT1 and HcMT2 increased as a function of Cu concentration increased. Maximum 300 folds of HcMT1 accumulation was observed at 320 µM. In case of HcMT2, the maximum accumulation was recorded at 320 µM where it showed almost 100 times higher than the control. In response to Cd, transcription of HcMT2 increased up to a maximum of 21 µM and decreased thereafter. HcMT1 expression was not induced by any of the concentrations of Cd used. Zn, Pb and Ni metals were not induced expression of HcMT1 or HcMT2 genes. The present results show that both MTs are having a particular pattern of regulation. HcMT1 is very sensitive to Cu induction and is probably involved in the detoxification of this metal. HcMT2 has a more pleiotropic role as it involved in Cu and Cd detoxification.

To gain more information about putative role of HcMT1 and HcMT2 genes in oxidative stress, the effect of H2O2 on both the genes were analyzed. Fifteen days old H. cylindrosporum was treated with 25 mM of H2O2 for different time intervals 0, 12, 24, 36, 48, 60 and 72 hours. Expression levels of both HcMT1 and HcMT2 were increased in presence of oxidative stress. The maximum induction was observed 48 hours and 36 hours for HcMT1 and HcMT2 respectively. The induction levels were not high compared to the expression levels under metal stress, suggesting the expression of HcMT1 and HcMT2 is more specific in response to metals than oxidative stress.

To characterize HcMT genes further, both genes were expressed in two Saccharomyces cerevisiae mutant strains, which are unable to grow on high concentration of metals. Two copper-sensitive strains cup1s and cup1∆ and one cadmium sensitive yap1 mutant were used. The yeast vector p424-HcMT1 or p424-HcMT2 was constructed under the transcriptional control of the yeast GPD (glyceraldehyde- 3-phosphate dehydrogenase) promoter. Vector p424 and the construct p424-HcMT1 and p424-HcMT2 were introduced into cup1∆ and yap1 cells using lithium acetate procedure and transformed cells were selected on appropriate complete synthetic medium (SD). For the functional complementation experiments, cultures of cup1∆ and yap1 yeast cells carrying either p424 or p424-HcMT1 and p424-HcMT2 were spotted on SD supplemented with 150 M CuSO4 and 40 M CdSO4 plates. Plates were incubated for 3 days at 30oC and photographed. In parallel experiments, Falcon jars containing 20 ml of fresh SD-Trp-Ura and SD-Ura media were inoculated with mid-log precultures of cup1∆ and yap1 cells containing p424, p424-HcMT1 and p424-HcMT2 to attain a starting optical density of 0.02 at 600nm. Cells were grown at 30oC and 220 rpm and CuSO4 (150 M) and CdSO4 (40 M) were added 5 hours after inoculation. The optical densities of the cultures were measured at 3 hour interval for 42 hours.

Complementation studies indicated that p424-HcMT1 and p424-HcMT2 harbouring cup1∆ cells were able to grow on 150 µM copper supplemented media, where as p424 transformed DTY4 cells growth were restricted. yap1 transformed with p424 vector was unable to grow at 40 µM cadmium, whereas the Cd-sensitive phenotype of the yap mutant was fully complemented by HcMT1 and HcMT2. Further, the restoration of Cd and Cu tolerance by expression of HcMT1 and HcMT2 were confirmed in liquid culture assays. The p424-HcMT1 and HcMT2 cup1∆ and yap1 cells grew well in medium containing 150 M CuSO4 and 40 M. These results highlighted the key role of HcMT genes in Cu and Cd detoxification.

The present study results show that H. cylindrosporum encodes two different metallothionein genes and each of them have a particular pattern of regulation which are specifically induced by Cu and Cd respectively. Altogether, these results show that the identification and functional verification of different MTs in ectomycorrhizal fungi and the study of their regulation is a prerequisite for a better understanding of heavy metal tolerance of these fungi and subsequently for the determination of their ability to improve heavy metal tolerance of their host plant.