EpABC Genes in the Adaptive Responses of Exophiala pisciphila to Metal Stress: Functional Importance and Relation to Metal Tolerance

Abstract

Exophiala pisciphila is one of the dominant dark septate endophytes (DSEs) colonizing metal-polluted slag heaps in southwest China. It shows numerous super-metal-tolerant characteristics, but the molecular mechanisms involved remain largely unknown. In the present study, the functional roles of a specific set of ATP-binding cassette (ABC) transporters in E. pisciphila were characterized. In total, 26 EpABC genes belonging to 6 subfamilies (ABCA to ABCG) were annotated in previous transcriptome sequencing libraries, and all were regulated by metal ions (Pb, Zn, and Cd), which was dependent on the metal species and/or concentrations tested. The results from the heterologous expression of 3 representative EpABC genes confirmed that the expression of EpABC2.1, EpABC3.1, or EpABC4.1 restored the growth of metal-sensitive mutant Saccharomyces cerevisiae strains and significantly improved the tolerance of Arabidopsis thaliana to Pb, Zn, and Cd. Interestingly, the expression of the 3 EpABC genes further altered metal (Pb, Zn, and Cd) uptake and accumulation and promoted growth by alleviating the inhibitory activity in yeast and thale cress caused by toxic ions. These functions along with their vacuolar location suggest that the 3 EpABC transporters may enhance the detoxification of vacuolar compartmentation via transport activities across their membranes. In conclusion, the 26 annotated EpABC transporters may play a major role in maintaining the homeostasis of various metal ions in different cellular compartments, conferring an extreme adaptative advantage to E. pisciphila in metal-polluted slag heaps. IMPORTANCE Many ABC transporters and their functions have been identified in animals and plants. However, little is known about ABC genes in filamentous fungi, especially DSEs, which tend to dominantly colonize the roots of plants growing in stressed environments. Our results deepen the understanding of the function of the ABC genes of a super-metal-tolerant DSE (E. pisciphila) in enhancing its heavy metal resistance and detoxification. Furthermore, the genetic resources of DSEs, e.g., numerous EpABC genes, especially from super-metal-tolerant strains in heavy metal-polluted environments, can be directly used for transgenic applications to improve tolerance and phytoextraction potential.

Physiological response mechanism of heavy metal‐resistant endophytic fungi isolated from the roots of Polygonatum kingianum

“…Metal detoxification and tolerance mechanisms of microorganisms often include uptake‐reduced, elimination‐enhanced, and intravesicular chelation with metal‐binding proteins and peptides, of which the process is regulated by a rich and complex population of genes (Guo et al, 2016; Priyadarshini et al, 2021). The expression of some uptake‐ and transport‐related proteins is particularly important for fungal HM detoxification, for example, the ATP‐binding cassette transporter EpABC2.1 of E. pisciphila , multidrug, toxic compound extrusion protein MTE1 of Tricholoma vaccinum and major promoter superfamily permease C17235 of Pisolithus albus , which can confer stronger Cd/Cu/Ni tolerance in transformants of Saccharomyces cerevisiae (Ali et al, 2012; Cao et al, 2019; Majorel et al, 2014; Schlunk et al, 2015). However, the molecular mechanisms underlying HM tolerance in fungi remain unclear and will be the focus of future experiments.…”

Plant association with dark septate endophytes: When the going gets tough (and stressful), the tough fungi get going

“…To avoid the intracellular accumulation of HM, DSEs can regulate the expression of metal ions transporters and compartmentalize ions through vesicle membrane fusion (Su et al, 2021;Zhao et al, 2015). Upon exposure to Pb and Zn, Exophiala pisciphila (Chaetothyriales) showed a down-regulation of its membrane transporters for the uptake of Zn 2+ , Fe 2+ and Ca 2+ , and up-regulation of transporters involved in the efflux of HM ions from the cytosol, outside a cell or inside vacuoles (Cao et al, 2019;Zhao et al, 2015). Exposure of E. pisciphila and Falciphora oryzae to Cd have revealed the sequestration of Cd 2+ and Zn 2+ in vacuoles and chlamydospores (Liu et al, 2021;Su et al, 2021), as a consequence of the over-expression of SNARE protein Syntaxin 1 involved in chlamydospore formation and vacuole enlargement (Su et al, 2021).…”

Transcriptome Analysis and Expression Profiling of Molecular Responses to Cd Toxicity in Morchella spongiola

“…ABC transporters, as one of the largest families of microbial membrane proteins, shows great important in resisting virulence and drug, especially in the function of maintaining metal ion homeostasis by transporting metal ions across the cell membrane in ionic/complex forms [20,21]. It was reported that the expression of 3 ABC genes (EpABC2.1, EpABC3.1, or EpABC4.1) of dominant dark septate endophytes (DSEs) could promote the restoring growth of metal-sensitive mutant by alleviating the inhibitory activity in yeast caused by Pb, Zn and Cd, and further altered toxic ions uptake and accumulation [27]. A specific ABC-transporter of Escherichia coli could mediate the entry of the Fe 2þ bounding siderophores through the inner membrane of the cell from the outer membrane under the help of FepB and FhuD chaperones [28].…”