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Published in final edited form as: Fungal Genet Biol. 2019 Jul 17;132:103253. doi: 10.1016/j.fgb.2019.103253

Epigenetic mechanisms of drug resistance in fungi

Zanetta Chang 1, Vikas Yadav 1, Soo Chan Lee 2, Joseph Heitman 1,#
PMCID: PMC6858951  NIHMSID: NIHMS1534953  PMID: 31325489

Abstract

The emergence of drug-resistant fungi poses a continuously increasing threat to human health. Despite advances in preventive care and diagnostics, resistant fungi continue to cause significant mortality, especially in immunocompromised patients. Therapeutic resources are further limited by current usage of only four major classes of antifungal drugs. Resistance against these drugs has already been observed in pathogenic fungi requiring the development of much needed newer antifungal drugs. Epigenetic changes such as DNA or chromatin modifications alter gene expression levels in response to certain stimuli, including interaction with the host in the case of fungal pathogens. These changes can confer resistance to drugs by altering the expression of target genes or genes encoding drug efflux pumps. Multiple pathogens share many of these epigenetic pathways; thus, targeting epigenetic pathways might also identify drug target candidates for the development of broad-spectrum antifungal drugs. In this review, we discuss the importance of epigenetic pathways in mediating drug resistance in fungi as well as in the development of anti-fungal drugs.

Keywords: Epimutation, Long non-coding RNA, Histone modification, Antifungal drug, Candida albicans, Mucor

1. Fungal diseases and antifungal resistance

Fungal diseases impact species across the plant and animal kingdoms, influencing food security, plant and wildlife extinctions, and human health worldwide (Brown et al., 2012; Fisher et al., 2012). Fungal infections are estimated to affect more than one billion people, with an estimated 150 million people suffering from severe or life-threatening forms of the disease (Bongomin et al., 2017). Furthermore, the overall incidence of fungal infections appears to be increasing, with newly emerging pathogens affecting all kingdoms of life including humans.

One of the factors contributing to the severity of fungal infections is the rapid emergence of antifungal drug resistance. There are several barriers to the effective treatment of fungal infections with antifungal drugs. First, the antifungal drugs available to treat human fungal pathogens are limited, with only four major classes of drugs clinically available: azoles, polyenes, echinocandins, and a nucleotide analog (McCarthy et al., 2017; Roemer and Krysan, 2014). Secondly, there is overlap between the antifungal agents employed to counter human and plant fungal infections. Similar mechanisms operate in both plant and human fungal pathogens to drive drug resistance (Fisher et al., 2018). Moreover, human fungal pathogens can also acquire azole-resistance by being exposed to agricultural azoles (Berger et al., 2017). Finally, many species of fungi are intrinsically resistant to some antifungal classes, and the mechanisms underlying this resistance have yet to be fully elucidated (Cowen et al., 2014; Perlin et al., 2017).

Acquired antifungal resistance has been studied across many important pathogens, and mechanisms of resistance have been identified for all four classes of antifungals. These include several well-studied genetic mechanisms, including mutations, aneuploidy, upregulation of stress response pathways, and biofilm formation (Cowen et al., 2014; Perlin et al., 2017; Robbins et al., 2017). However, the study of epigenetic pathways is beginning to shed new light on the role of epigenetic factors in already existing or novel mechanisms through which fungi can develop antifungal resistance.

2. Epigenetic mechanisms in fungi

Epigenetics refers to cellular changes mediated by factors other than DNA sequence modifications. These changes do not alter DNA sequences or protein coding but instead transiently affect the expression of target genes. Broadly, epigenetic modifications can be categorized into two main mechanisms: RNA-based and chromatin-based.

RNA-based pathways include RNA interference (RNAi) and non-coding RNAs. RNAi is a mechanism mediated by small RNAs (sRNAs) produced through a core RNAi pathway involving RNA-dependent RNA polymerases and the endonuclease Dicer (Villalobos-Escobedo et al., 2016). Processed sRNAs are then incorporated into the Argonaute complex, which selectively targets complementary RNAs and either induces degradation or inhibits translation of the target RNA. In some instances, the RNAi machinery can also recruit heterochromatin proteins to target genes, thus inhibiting gene expression (Martienssen and Moazed, 2015).

Long non-coding RNAs (lncRNAs) are defined as RNA molecules that are generally larger than 200 bp, which distinguishes them from small nuclear RNAs (Carninci et al., 2005). Additionally, most small nuclear RNAs are transcribed by RNA polymerase III, whereas lncRNAs are transcribed by RNA polymerase II. Many lncRNAs also undergo RNA processing similar to mRNAs, including 5’ capping, splicing, and polyadenylation. lncRNAs comprise a high percentage of total RNA transcripts; for example, in the human genome, the number of lncRNAs is ˜4-fold higher than that of gene-coding RNAs (Kapranov et al., 2007). lncRNAs are mainly found in the nucleus and are subjected to degradation by exosomes. Therefore, they have previously been considered transcriptional noise (Struhl, 2007) However, several studies have revealed that lncRNAs play roles in epigenetic gene regulation (Dimond and Fraser, 2013; Mikkelsen et al., 2007; Moran et al., 2012).

Chromatin modifications consist of mechanisms that alter chromatin either chemically or structurally (Allis and Jenuwein, 2016; Chen et al., 2017). Chemical modifications include posttranslation modifications (PTMs) of histone proteins and DNA methylation whereas structural changes include chromatin remodeling and DNA-DNA interactions. The N-terminal tail of histones in nucleosomes provides a substrate for a number of PTM events (Bannister and Kouzarides, 2011). These include alkylation (methylation and acetylation), phosphorylation, ubiquitination, and sumoylation, among others. These modifications lead to changes in chromatin organization that allow selective accessibility for transcription factors to specific genomic regions while restricting the binding of transcription machinery to other genomic locations. Examples of these epigenetic modifications are listed below.

  1. DNA methylation: A methyl group is added to the cytosine bases of DNA, giving rise to 5-mC base modification (Chen et al., 2017). This modification is abundant in the human genome as well as in some fungi. Methylation of adenine bases also occurs in some organisms and can play critical roles (Parashar et al., 2018).

  2. Histone modifications: Histones are the core components of nucleosomes and act as substrates for several PTMs (Bannister and Kouzarides, 2011; Zhao and Garcia, 2015). Well-characterized PTMs include methylation, acetylation, and phosphorylation. While some of these modifications are stable, most are dynamic. Additionally, the presence of one PTM could restrict the introduction of others on the same or neighboring residues, most likely due to steric constraints. The enzymes that mediate these modifications include lysine acetyltransferases (KAT), histone methyltransferases (HMT), and protein arginine methyltransferases (PRMT), whereas enzymes that remove these modifications include lysine deacetylases (KDAC) and lysine demethylases (KDM) (Marmorstein and Trievel, 2009). The KATs and KDACs specifically modifying histones are named as HATs and HDACs, respectively. Histone modifications can alter gene expression levels by allowing or restricting binding of transcription factors, enhancers, or chromatin remodeling proteins. For example, acetylated histones lead to an open conformation promoting transcription, whereas deacetylated histones render the chromatin compact, thus inhibiting transcription. Both HDAC and HATs catalyze reversible reactions and thus dynamically regulate transcription (Bannister and Kouzarides, 2011; Clapier and Cairns, 2009; Grunstein, 1997).

  3. Chromatin remodeling: Chromatin is a highly dynamic structure, in contrast to previous hypotheses that suggested it to be either euchromatin (loosely packed and transcribed) or heterochromatin (tightly packed and nontranscribed). This dynamic property allows chromatin to change nucleosome positioning in the genome, a critical factor required for transcription initiation (Clapier and Cairns, 2009). The process of chromatin remodeling is an active process and requires ATP-dependent nucleosome remodelers (Clapier et al., 2017). Two well-studied chromatin remodelers include the ISWI and SWI/SNF proteins. Another critical factor that contributes to chromatin compaction or loosening is the looping of DNA sequences, providing 3-D interactions that can further influence transcription. A well-studied example of such an interaction is enhancer-promoter communication that can recruit transcription factors to initiate transcription.

Regardless of whether epigenetic modifications are RNA- or chromatin-based, they provide an additional route via which organisms can rapidly respond to changing environments. Epigenetic modifications can be heritable, but they are also transient and rapidly reversible. Thus, they are more flexible than direct changes to the genetic code. Epigenetic modifications serve as a bet-hedging strategy by which fungi can increase their phenotypic plasticity to respond to environmental cues and stresses, including drug stress.

3. RNA-based forms of epigenetic antifungal resistance

Two RNA-based mechanisms of epigenetic antifungal resistance have been identified in fungi: epimutation and chromatin silencing directed via long non-coding RNAs (lncRNAs).

Epimutation, a novel form of RNAi-based epigenetic drug resistance, was first described in Mucor circinelloides. M. circinelloides is a fungus, belonging to the Mucorales, with well-characterized RNAi pathways, both canonical and non-canonical, which can induce silencing of both endogenous and exogenous genetic elements (Torres-Martinez and Ruiz-Vazquez, 2016). Epimutation in fungi was discovered as a mechanism conferring drug resistance to the antifungal agents FK506 and rapamycin (Calo et al., 2014). Both of these drugs bind to the peptidyl-prolyl isomerase FKBP12 and then inhibit calcineurin and TOR, respectively. Therefore, resistance to these agents can be induced through mutation or loss of FKBP12, encoded by the fkbA gene.

Epimutation was identified in M. circinelloides strains that became FK506 resistant through the endogenous expression of sRNAs against the fkbA gene, leading to degradation of fkbA mRNA (Figure 1A). sRNA sequencing of epimutant strains revealed that both sense and antisense sRNAs were present, and mapped to the fkbA locus without spreading to surrounding regions. Epimutation in these strains was transient and reversible; after several passages on standard laboratory media without antifungal exposure, epimutant strains ceased to produce sRNAs corresponding to fkbA and reverted to a wild-type level of drug sensitivity. Selection of FK506-resistant isolates from a reverted epimutant strain again yielded epimutants at the same original frequency, showing that the process was strictly epigenetic.

Figure 1. Modes of epigenetic-mediated drug resistance in fungi.

Figure 1.

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(A) RNAi-mediated epimutations are induced in the drug resistane cells where the mRNAs of the target gene are degraded allowing the cells to grow in drug containing media. How mature mRNA are targeted by the RNAi machinery may involve anti-sense transcripts generated by RdRP. (B) Non-coding RNAs (ncRNAs) might repress transcription of specific genes allowing cells to grow in the presence of a drug. One of the known mechanisms includes ncRNA-mediated genome condensation, which restricts the binding of transcription factors at the promoter of the target gene. (C) KDACs (including HDACs) and KATs (including HATs) can modulate expression levels of target genes by (i) opening or closing the chromatin or (ii) regulating the activity of chaperones like Hsp90, both of which can affect multiple stress-related responses.

It was subsequently demonstrated that epimutation in M. circinelloides can confer resistance to additional antifungal agents. Resistance against the antifungal agent 5-fluoroorotic acid (5-FOA) is well characterized in other fungi and involves loss of function mutation in either the pyrF (URA5 in other fungi) or the pyrG (URA3) genes. BothpyrF and pyrG encode enzymes in the pyrimidine biosynthetic pathway that convert 5-FOA from an inactive prodrug into a toxic nucleotide analog (Boeke et al., 1984; Chang et al., 2019). Epimutation in M. circinelloides is capable of inducing transient resistance to 5-FOA through the accumulation of sense and antisense sRNAs by silencing either the pyrF or pyrG loci. This demonstration that epimutation can drive resistance to a second class of antifungal agent suggests that epimutation is not limited to a specific genetic locus or antifungal mechanism. Thus, it was hypothesized that epimutation is not only a novel form of antifungal resistance but also serves as a general mechanism through which M. circinelloides can generate phenotypic plasticity and rapidly adapt to environmental stresses. Epimutation mediated drug resistance in other fungi as well as in environmental samples remains to be explored. Epimutations have been described in several human diseases and in plant flower pattern mutants; in these cases epimutation results from DNA methylation rather than RNAi (Horsthemke, 2006; Martin et al., 2005; Zoghbi and Beaudet, 2016).

The process of epimutation in M. circinelloides is carried out by RNAi machinery components from the canonical RNAi machinery. The M. circinelloides genome contains multiple copies of all three canonical RNAi components – RNA-dependent RNA polymerase (RdRP), dicer (Dcl), and argonaute (Ago). The function of Dcl1, Dcl2, Ago1, and RdRP2 are all essential for the process of epimutation, as loss-of-function mutation in the genes encoding these factors abolishes the ability to develop epimutation-derived drug resistance (Calo et al., 2012; Calo et al., 2014; Cervantes et al., 2013; de Haro et al., 2009; Nicolas et al., 2010). Further work identified two additional components of the epimutation pathway, quelling induced protein (Qip1) and a Sad3-like helicase, and demonstrated that the epimutation pathway competes with an alternative RNA-dependent RNA polymerase-dependent, Dicer-independent RNA degradation pathway (Calo et al., 2017; Trieu et al., 2015). Mutation of the components of this alternative pathway - such as the RNA polymerases RdRP1 or RdRP3, or the RNase III-like protein R3B2 - leads to increased rates of epimutation, potentially due to an increased availability of the target RNA for the epimutant pathway. The RNAi machinery requires a double-stranded RNA as substrate, thus raising a question about how a specific double-stranded RNA is produced against the drug-resistant genes. The original study suggested two models for this phenomenon, one of which suggested that sRNAs are produced constitutively at a low level against the entire genome (Calo et al., 2014). While some evidence supports this hypothesis in M. circinelloides, future studies will shed more light on this process (Nicolas et al., 2010). Additionally, RNAi is present in many fungal species where in most cases it acts to suppress transposition (Villalobos-Escobedo et al., 2016). It will be interesting to see if RNAi also leads to sRNA production from genes in species in which it has not yet been detected yet.

A recent study demonstrated that lncRNAs can regulate drug tolerance in Schizosaccharomyces pombe (Ard et al., 2014). Deletion of a lncRNA, ncRNA.1343, resulted in hypersensitivity to a broad spectrum of drugs, including the microtubule-depolymerizing agent thiabendazole, the DNA synthesis inhibitor hydroxyurea, and the cAMP synthesis inhibitor caffeine. Further investigation revealed that the ncRNA.1343 controls the neighboring tgp1 gene, which encodes a glycerophosphodiester transporter 1 (Tgp1) (Figure 1B). The lncRNA increases nucleosome density to limit access for transcription of the tgp1 gene. Thus, the lncRNA regulates drug resistance through transcriptional interference as the deletion of the lncRNA induces expression of tgp1, rendering the cell drug-sensitive (Ard et al., 2014).

Even among evolutionarily closely related species, lncRNAs are in most cases divergent. However, ncRNA.1343 neighboring the tgp1 gene is one of eight lncRNAs conserved in three different Schizosaccharomyces species (Ard et al., 2014; Rhind et al., 2011). Thus, this lncRNA mechanism appears to be a conserved regulatory circuit. It will be of interest to determine if this type of lncRNA-associated drug sensitivity system is conserved in other fungal systems. Overall, identification of lncRNAs and their roles in epigenetic regulation of cellular processes in fungi has been understudied compared to humans and other mammals (Carninci et al., 2005; Iyer et al., 2015; Till et al., 2018). More investigation is required to determine whether lncRNAs influence antifungal drug resistance in fungi other than S. pombe, especially in multi-drug-resistant pathogenic fungi such as Candida auris.

4. Chromatin-based forms of epigenetic resistance

Addition of a methyl or alkyl group to histone proteins is a major form of chromatin modification present among many organisms. These modifications can modulate gene expression levels by either altering chromatin structure or recruiting specific proteins. Thus, these modifications can have a considerable impact on the expression of resistance genes without changing the DNA sequence. However, the potential of chromatin modifiers in regulating antifungal drug resistance is not well explored.

Histone acetylation has been shown to be involved in the development of antifungal resistance in Candida albicans (Garnaud et al., 2016; Li et al., 2015; Robbins et al., 2012). Genes involved in the deacetylation process, HDA1 and RPD3, were found to be expressed at a higher level in azole-resistant cells of C. albicans. Interestingly, expression decreased once resistance was established, indicating that the genes play a transient but crucial role in this process. This effect was attributed to the stability and function of heat shock protein (Hsp90) protein in the absence of deacetylating enzymes (Cowen and Lindquist, 2005; Garnaud et al., 2016; Li et al., 2015; Robbins et al., 2012) (Figure 1C). Another study reported that depletion of H3K56 acetylation renders C. albicans cells hypersensitive to echinocandins, but not to fluconazole (Wurtele et al., 2010). Additionally, changes in H3K56 acetylation, either genetically or pharmacologically, reduce C. albicans virulence in murine models. Another deacetylase complex, (comprised of Set3, Hos2, Snt1, and Sif2), was shown to mediate drug resistance in C. albicans biofilms, which are resilient, surface-attached communities of cells that enable C. albicans to evade various drugs and colonize the human body (Nobile et al., 2014). Deletion of HDAC genes in Cryptococcus neoformans resulted in reduced pathogenicity as well as sensitivity to multiple stress conditions (Brandao et al., 2018). One of the HDACs, Hda1, was shown to be regulating the genes required for virulence and stress adaptation. Multiple studies in Aspergillus and plant pathogenic fungi also demonstrated the role of histone deacetylases in virulence and growth (Lamoth et al., 2015) (Figure 1C).

In addition to histone deacetylases, histone acetyltransferases and chromatin remodelers also play essential roles in drug resistance and biofilm formation. One such acetyltransferase complex, NuB4, was shown to be required for stress resistance (Tscherner et al., 2015). Deletion of the regulatory subunit of this complex also led to increased antifungal drug tolerance. Overall, these results suggest that histone deacetylation plays a role in conferring drug resistance. While the exact mode of action for most of these deacetylases remains to be elucidated, studies in both Candida and Aspergillus suggested a role for Hsp90 in this process (Cowen and Lindquist, 2005; Lamoth et al., 2015). Deacetylation of Hsp90 is necesssary for its proper interaction with client proteins such as calcineurin, which in turn governs stress responses including drug resistance (Robbins et al., 2012). The Swi/Snf chromatin remodeling complex of C. albicans is involved in fluconazole resistance through alterations in Mdr1 expression. The Swi/Snf chromatin remodeling complex acts as a coactivator for Mrr1, the transcription factor that enhances the expression of Mdr1, a drug efflux pump (Liu and Myers, 2017). Deletion of Snf2 significantly reduces the increase in Mdr1 expression, making it more susceptible to the drug.

An analogous example of histone modification leading to epigenetic drug resistance occurs in the malaria parasite, Plasmodium falciparum. During the asexual blood cycle of the parasite, chromatin-based epigenetic silencing is required for switching between copies of clonally variant gene families, including those encoding erythrocyte membrane protein (PfEMP1), a major surface antigen, and clag3, a channel protein involved in erythrocyte invasion (Cortes et al., 2007;Cortes et al., 2012). Silencing of clag3 was associated with resistance to the antimalarial agent blasticidin S due to low uptake of the drug (Sharma et al., 2013). Further work suggested that other antimalarial drugs may also be taken up via clag3, such that clag3 silencing may induce multi-drug resistance (Mira-Martinez et al., 2019). P. falciparum repression of these clonally variant gene families is mainly mediated by methylation of histone 3 (H3K9me3), whereas activation is mediated by acetylation (H3K9ac). A role for ncRNA in this regulation has been proposed and future studies will provide further insight into this process (Cortes et al., 2012).

5. Epigenetic pathways as potential antifungal drug targets

Epigenetics plays a role in drug resistance in pathogenic fungi. It is, therefore, possible that these mechanisms can be targeted or utilized to develop novel therapies against fungal diseases. Epigenetic pathways are under investigation for the development of anti-cancer drugs, some of which have been FDA approved for cancer chemotherapy (Heerboth et al., 2014; Ivanov et al., 2014). However, research targeting these pathways to develop anti-fungal drugs is less advanced. One factor could be a limited understanding of these pathways in fungi. Additionally, some epigenetic pathways are absent in fungi. For example, several fungi, including pathogenic Candida species, have lost functional RNAi machinery (Billmyre et al., 2013; Nicolas and Garre, 2016). Interestingly, the loss of RNAi has been proposed to play roles in pathogenesis, however the mechanisms are not yet understood (Billmyre and Heitman, 2017). Increased mutation rates providing better adaptation, reduction of the genome leading to faster replication, or the occurrence of dsRNA viruses are among the mechanisms that might be contributing to increased pathogenesis in RNAi-deficient fungi (Billmyre and Heitman, 2017; Yadav et al., 2018). Among fungi, RNAi is well understood in the fission yeast, Schizosaccharomyces pombe, which is non-pathogenic and harbors species-specific essential RNAi proteins (Martienssen and Moazed, 2015). Studies in C. neoformans also revealed species-specific novel RNAi components, but whether they play a role in virulence remains to be explored (Burke et al., 2019; Dumesic et al., 2013; Feretzaki et al., 2016).

While targeting novel and fungal-specific RNA pathway genes is one possible option to target epigenetic pathways, RNAi also provides a unique mechanism to specifically repress drug resistance genes and thus reduce the chances for fungal resistance. A proof of concept study in A. nidulans showed that RNAi can be used to specifically repress genes (Kalleda et al., 2013). More recently, studies in plant pathogenic fungi Fusarium graminearum and Colletotrichum acutatum revealed that hairpin RNAs can be transfected into fungi repressing target genes, or dsRNA can be sprayed on a host plant thus killing the associated fungal pathogen (Koch et al., 2016; Mascia et al., 2014). However, several challenges remain before this approach can be employed more widely. Some of these challenges include the stability of hairpin structures and efficient and specific delivery of double-stranded RNA to the fungi during infections.

The deletion of multiple HATs and HDACs has been shown to affect virulence and filamentation of several fungal species (Garnaud et al., 2016; Lopes da Rosa and Kaufman, 2012). In comparison to human HATs and HDACs, some fungal counterparts show significant differences in their own sequence or their substrate sequences. An evolutionary conserved HAT, Hat1, recognizes a specific set of lysine residues that are either missing or are not modified in humans (Kuchler et al., 2016). Similarly, RdpA of A. nidulans harbors a fungal-specific conserved C-terminal motif that can be used as a drug target (Tribus et al., 2010). These differences make HAT/HDAC enzymes targets for development of new antifungal drugs. Several HDAC inhibitors were tested against Candida, Aspergillus and C. neoformans and exhibited antifungal activity (Brandao et al., 2015; Garnaud et al., 2016; Lamoth et al., 2015). A well-known broad-spectrum HDAC inhibitor, Trichostatin A (TSA), displays antifungal activity against Aspergillus and Trichophyton (Tsuji et al., 1976). TSA induces both the white-to-opaque transition and the yeast-hyphal dimorphic transition in C. albicans (Hnisz et al., 2010; Klar et al., 2001). TSA and sodium butyrate, another HDAC inhibitor, exhibit dose-dependent effect on virulence factors in C. neoformans and interfere with the mating process (Brandao et al., 2015). While HDAC inhibitors have little antifungal activity on their own, their action is enhanced in the presence of other antifungal agents. An HDAC inhibitor significantly increased azole activity against Candida species (Mai et al., 2007; Smith and Edlind, 2002). Similarly, TSA increased the antifungal activity of caspofungin, but not of voriconazole, against A. fumigatus (Lamoth et al., 2014). Another fungal-specific HDAC Hos2 inhibitor, MGCD290, has been developed and found to be active against many fungi (Pfaller et al., 2009), and to potentiate azole activity. Thus, HDAC inhibitors hold promise as monotherapy as well as in combination with existing antifungal drugs. Significant divergence of the proteins that are involved in histone acetylation/deacetylation further provides an advantage because this could significantly reduce side effects on host cells. HDAC inhibitors also have activity against parasites (Wang et al., 2015) and hence, could be used to treat multiple eukaryotic infectious diseases. Given that HDAC inhibitors are in clinical trials for anti-cancer activity, harnessing their activity against fungal pathogens might pave the way for the development of novel and more potent antifungal drugs.

Highlights (3-5 points, each with a limit of maximum 85 characters).

  • Epigenetic mechanisms produce dynamic, reversible modes of drug resistance in fungi.

  • Fungi show both RNA-based and chromatin-based modes of drug resistance.

  • Epimutations and long non-coding RNAs are major RNA-based forms of resistance.

  • Histone acetylation/deacetylation is involved in drug resistance development.

  • Epigenetic pathways can be broad-spectrum targets for antifungal drug development.

Acknowledgments

We thank Sheng Sun and Shelby Priest for critical reading of the manuscript. This work was funded by NIH/NIAID R37 MERIT award AI39115-21, R01 grant AI50113-15, P01 grant AI104533-05, and R01 grant AI112595-04 awarded to JH. SCL was supported by a Korean Food Research Institution (KFRI) and holds a Voelcker Fund Young Investigator Pilot Award from the Max And Minnie Tomerlin Voelcker Fund. We also thank CIFAR program, Fungal Kingdom: Threats & Opportunities for support.

Footnotes

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