**2. Fungal–Metal Interactions**

Metals play critical roles in fungal homeostasis. They are required for various biochemical processes, usually as enzymatic cofactors. Metals most recognized for their importance in fungi are copper, iron, zinc, and manganese. Pertaining to zinc, approximately 5% of fungal proteomes correlate to zinc-binding proteins, and 8% of yeast genomes correlate to zinc-binding proteins. In the model yeast *S. cerevisiae*, large portions of these zinc-binding proteins are related to critical functions, including DNA binding (31% of zinc-binding proteins), the regulation of transcription (25%), transcription factor activity (19%), and response to chemical stimuli (15%) [105,107,108]. Fungal–copper interactions are necessary for the activation of metalloproteins involved in biochemical processes. This includes the activation of superoxide dismutase, which is responsible for cellular detoxification of reactive oxygen species (ROS), virulence in pathogenic species, and activation of cytochrome *c* oxidate, a catalyst within the electron transport chain [39,48]. Iron is also essential for fungal virulence in pathogenic species, most importantly as an integral component of iron-sulfur clusters which are required for the activation of nuclear proteins involved in DNA repair [61]. Manganese also plays a critical role in fungi, in particular, in filamentous species where it (or copper) is required for the activation of manganese peroxidase. Dependent on nutrient availability, white-rot fungi utilize manganese peroxidase as a secondary metabolite to depolymerize lignin for nutrients; others are manipulated for increased manganese peroxidase production and extraction for use in the degradation of organo-pollutants [96,98].

Very few metals that are not considered essential have also been identified in some fungal–metal interactions; these include magnesium and molybdenum. Magnesium is a well-known micronutrient in other eukaryotic organisms, however, its homeostasis in fungi is undetermined. Only in recent years has magnesium been identified as a requirement for virulence in the agriculturally relevant fungus *Magnaporthe oryzae* [127]. Molybdenum is a metal that is discussed significantly less in eukaryotic homeostasis. It has only been identified as a cofactor for four human proteins, and in fungi it has only been suggested that it plays an unidentified role as a nitrate reductase and a xanthine dehydrogenase [128,129]. Other metals such as silver, gold, lead, nickel, and cadmium have only been implicated in fungal–metal interactions related to toxicity, nanoparticle myco-synthesis, and heavy metal myco-remediation, but information pertaining to homeostasis is limited [103,130,131].

#### *2.1. Zinc*

Zinc is a transition metal required for fungal survival and is necessary for various functions, including the structuring of nucleic acids, physical growth and, most predominately, protein folding [132,133]. In its role in DNA binding, zinc presents itself in class III zinc finger proteins, also known as zinc cluster proteins (Zn(II)2Cys6), found only in Ascomycetes (with the singular exception of *Lentinus edodes*) [107,134–136]. This protein class binds DNA, which is critical for the transcriptional activation and regulation of gene products [105,134].

In agriculture, fungal infections threaten food security by increasing global crop loss [137,138]. Traditionally, antifungal azoles have been used to combat disease, but with the emergence of azole-resistant pathogens, scientists have begun to develop possible alternatives, such as zinc-containing compounds [138,139]. Reports have demonstrated that zinc oxide nanoparticles (ZnO NPs) can control postharvest mold, plant wilts, and grey mold disease caused by *Aspergillus niger*, *Fusarium oxysporum*, and *Botrytis cinerea*, respectively [7,140–143]. It has also been demonstrated that ZnO NPs can significantly reduce the production of the mycotoxin fusaric acid from *F. oxysporum* [144]. This is significant because mycotoxins are common secondary metabolites of fungal pathogens with high rates of toxicity against cereal crops that can result in crop loss, and if consumed can result in a wide array of diseases in livestock [145,146]. Fusaric acid, in particular, can inhibit the production of dopamine-beta-hydroxylase, which acts as a messenger of signals within the nervous system and is responsible for altering the enzyme tyrosine

hydrolase, which is involved in a rate-limiting step in catecholamine synthesis [147–149]. Zinc perchlorate Zn(ClO4)<sup>2</sup> and zinc sulfate (ZnSO4) also inhibit mycelial growth that produces mycotoxins and reduces the production of mycotoxins themselves [150,151].

#### 2.1.1. Zinc Transport and Homeostasis

Many fungi have mechanisms of zinc transport similar to that of other eukaryotes, through the ZRT (zinc regulated transporter)-IRT (iron-regulated transporter)-like protein (ZIP) family and the cation diffusor facilitator (CDF) protein family [152,153]. In *S. cerevisiae,* zinc transport occurs through several protein groups; the ZIP protein family (via Zrt1, Zrt2, and Zrt3), the CDF protein family (via Zrc1, Cot1, and Msc2), the ferrous transport protein Fet4, and others (Figure 1) [105,107,108,133,154,155]. Zrt1 and Zrt2 are high and lowaffinity plasma membrane transporters, respectively; both *ZRT1* and *ZRT2* are upregulated in zinc-deficient conditions and repressed when zinc conditions are favorable [15,22,156]. In an excess-zinc environment, Zrc1 and Cot1 mediate zinc transport from the cytosol into the vacuole to prevent toxicity [20,28]. In a zinc-limiting environment, zinc is released back into the cytosol from the vacuole via Zrt3 or is scavenged by zincophore Zps1 [106,157,158]. Zap1 regulates the transcription of *ZPS1* and contains two activators, Ad1 and Ad2, either independently activated or inactivated by the direct binding of zinc ions [105,108,159,160]. These mechanisms effectively control intracellular zinc uptake and help prevent excess accumulation in *S. cerevisiae*.

In filamentous Ascomycota, such as *Apergillus fumigatus*, genes in the ZIP family (*zrfA*, *zrfB*, *zrfC*, *zrfD*, *zrfE* and *zrfH*) also regulate zinc transport [23,27,161]. *zrfA* and *zrfB*, orthologues of *S. cerevisiae ZRT1* and *ZRT2*, respectively, encode zinc membrane transporters that operate in acidic, low-zinc environments and are activated by transcription factor ZafA [161,162]. Conversely, the *zrfC* gene product is an alkaline zinc transporter activated in high pH, zinc-limiting conditions [23,27]. *zrfD/E/H* are not restricted by pH and can function in either acidic or alkaline environments [23]. In *F. oxysporum*, *zrfA* and *zrfB* are also zinc importers regulated by transcription factor ZafA [163]. During infection, ZafA allows *F. oxysporum* to adapt to a zinc-limiting environment, such as if the host enacts nutritional immunity to deprive it of this essential metal [163]. Basidiomycetes have similar homology. *Ustilago maydis* UmZRT1 and UmZRT2 genes, and *Cryptococcus neoformans* Zip1 and Zip2 are homologous to *S. cerevisiae ZRT1* and *ZRT2*, respectively, with similar transport function [20,24,26]. Similarities also exist in the prevention of zinc over-accumulation. *C. neoformans* Zrc1 is homologous to *S. cerevisiae* Zrc1 and mediates zinc transport into the vacuole to prevent toxicity and decrease zinc sensitivity [20].

Mechanisms of zinc uptake and transport in fungi are mostly conserved through *S. cerevisiae* ZIP proteins and homologs. The next section will discuss how negative homeostatic interventions can result in toxicity.

### 2.1.2. Zinc Toxicity

Zinc-based antifungal compounds have mechanisms of toxicity that vary between species. Zinc pyrithione (ZPT), is a zinc ionophore often used to treat fungal dandruff caused by *Malassezia* spp. and induces toxicity by increasing cellular zinc uptake [164,165]. ZPT also causes partial mitochondrial malfunction by inhibiting mitochondrial synthesis of iron-sulfur clusters, which are integral in electron transport, respiration, and DNA repair and replication [165,166]. In contrast to *Malassezia* spp., ZPT toxicity in *S. cerevisiae* is not a result of increased zinc uptake, rather of increased copper uptake which overloads homeostatic systems [164,167,168]. ZnO NPs are also being explored for their antifungal properties. In *S. cerevisiae*, ZnO NPs reduce ergosterol biosynthesis which, in turn, increases cellular leakage (up to 24%) and depolarization, reduces cell wall integrity, and increases the occurrence of ROS [83]. In filamentous fungi, mechanisms of toxicity are not wellstudied. In ericoid fungi, zinc ions reduced hyphal growth by increasing chitin deposition within the cell wall, preventing hyphal extension; zinc also increases hyphal branching and apical swelling, resulting in atypical hyphal morphology [88]. In the molds, excessive zinc

exposure reduces hyphal growth, alters hyphal morphology and interrupts conidia and conidiophore development, limiting reproductive capabilities [87]. Zinc sensitivity can aid in the reduction of fungal pests; however, the development of tolerance and resistance can be an impedance.
