Functional and transcript analysis of a novel metal transporter gene EpNramp from a dark septate endophyte (Exophiala pisciphila)

Abstract

Various metal transporters mediate sub-cellular sequestration of diverse metal ions, contribute to cellular metal tolerance, and control metal partitioning, particularly under conditions of high rates of metal influx into organisms. In the current study, a ubiquitous and evolutionary conserved metal transporter gene, homology to natural resistance associated macrophage protein (Nramp), was cloned from a metal-tolerant isolate of dark septate endophyte (DSE, Exophiala pisciphila), and its functional and transcript characterization were analyzed. The full-length Nramp gene from E. pisciphila (named EpNramp) was 1716 bp and expected to encode a polypeptide of 571 amino acid residues. EpNramp fused to green fluorescent protein suggested that EpNramp was a plasma membrane metal transporter, which was consistent with the results of bioinformatics analysis with 11 transmembrane domains. Yeast functional complementation revealed that EpNramp could complement the growth defect of Fe-uptake yeast mutant (fet3fet4 double mutant) by mediating the transport of Fe2+. Expression of EpNramp increased Cd2+ sensitivity and Cd2+ accumulation in yeast. In addition, qPCR data revealed that E. pisciphila significantly down-regulated EpNramp expression with elevated Cd2+ exposure. Altogether, EpNramp is a bivalent cation transporter localized in cell membrane, which is necessary for efficient translocation of both Fe and Cd, and its activities partly attributed to the tolerance of DSE to toxic and excessive Cd2+ supplements.