Fungal Genetics and Biology 66 (2014) 11–18

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Review

Molecular mechanisms of Aspergillus flavus secondary metabolism and development Meareg G. Amare, Nancy P. Keller ⇑ University of Wisconsin – Madison, 3476 Microbial Sciences Building, 1550 Linden Drive, Madison, WI 53706-1521, United States

a r t i c l e

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Article history: Received 3 January 2014 Accepted 25 February 2014 Available online 5 March 2014 Keywords: Aspergillus flavus LaeA VeA Aflatoxin Gene cluster Quorum

a b s t r a c t The plant and human opportunistic fungus Aspergillus flavus is recognized for the production of the carcinogen aflatoxin. Although many reviews focus on the wealth of information known about aflatoxin biosynthesis, few articles describe other genes and molecules important for A. flavus development or secondary metabolism. Here we compile the most recent work on A. flavus secondary metabolite clusters, environmental response mechanisms (stress response pathways, quorum sensing and G protein signaling pathways) and the function of the transcriptional regulatory unit known as the Velvet Complex. A comparison to other Aspergilli reveals conservation in several pathways affecting fungal development and metabolism. Ó 2014 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. flavus genome: secondary metabolite gene clusters . . . . . . . . . . . . . Global regulation by the Velvet Complex . . . . . . . . . . . . . . . . . . . . . . . . Quorum sensing in A. flavus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conserved Aspergillus proteins involved in morphogenesis in A. flavus Oxidative stress response in secondary metabolism and development Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Aspergillus flavus is a ubiquitous saprophytic fungus found in soils across the world. Although first described in 1809, A. flavus was thrown into the limelight in 1962 as a result of the Turkey X disease that killed thousands of poultry (Nesbitt et al., 1962). The Turkey X outbreak led to the discovery of aflatoxin, a fungal mycotoxin that had contaminated the poultry feed. Since then, A. flavus and aflatoxin have had tremendous economic and health impacts across the world (Amaike and Keller, 2011). Although aflatoxin is noted as the primary metabolite causing human disease, the ⇑ Corresponding author. E-mail address: [email protected] (N.P. Keller). http://dx.doi.org/10.1016/j.fgb.2014.02.008 1087-1845/Ó 2014 Elsevier Inc. All rights reserved.

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11 12 14 15 15 15 16 16 16

fungus produces several toxic metabolites that may also contribute to ill health as covered below. A. flavus survives as conidia or sclerotia in soil and organic debris. While conidia allow the fungus to mass-disseminate, sclerotia enable survival in harsh environmental conditions and can germinate once conditions improve. On host tissue, including that of humans, animals and plants, conidia germinate and grow as mycelia which can develop into either conidiophores or sclerotia depending on environmental and nutritional cues. While the conidium is considered the predominant infectious spore, the predominant reproductive form in soil is not known. Sclerotia contain the sexual ascospores of the fungus, which until recently were only reported to occur in experimental laboratory conditions but have now been reported to occur in the field also (Horn et al., 2009, 2013).

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A. flavus colonizes and produces aflatoxins (there are several forms of aflatoxin with aflatoxin B1 being the most carcinogenic) in oil-rich agricultural crops including maize, peanuts and cottonseeds both pre- and post-harvest. As aflatoxin is toxigenic as well as carcinogenic, controlling aflatoxin contamination of crops is vital. However, controlling contamination, both pre- and postharvest, induces tremendous monetary losses worldwide. In the US, aflatoxin contamination incurs economic losses of approximately US $1 billion per annum (Vardon et al., 2003) while African countries lose approximately $670 million from failure to meet European export standards (Otsuki et al., 2001). In an effort to curb aflatoxin exposure, the US Food and Drug Administration only allows 20 ppb in food and 0.5 ppb in milk (Georgianna and Payne, 2009) while some countries such as those in Europe have even stricter guidelines. On the other hand, developing countries often have lax, if any, guidelines for aflatoxin contamination. Aflatoxins have a wide range of health impacts depending on the aflatoxin dose. Acute aflatoxicosis, arising from high-dose aflatoxin intake over a short period, often results in aflatoxinpoisoning outbreaks killing scores of people. The quintessential examples for this are the recurrent outbreaks seen in the East African country Kenya, which experienced its worst outbreak in 2004 with 317 cases and 125 reported deaths (Azziz-Baumgartner et al., 2005). Chronic aflatoxicosis, arising from low-dose aflatoxin consumption over an extended period, can result in immune suppression, stunting and liver cancer. Aflatoxin-induced liver cancer is known to arise from a mutation in the tumor suppressor gene, p53, in the liver (Hsu et al., 1991). Perhaps exacerbating the problem is the fact that exposure to aflatoxin B1 in hepatitis B virus-endemic areas highly increases the chances of developing hepatocellular carcinoma by as much as 30-fold, creating severe health problems in developing countries where both are common occurrences (Groopman et al., 2008). To a lesser extent, aflatoxin B1 and hepatitis C virus also exhibit a similar relationship leading to increased chances of developing hepatocellular carcinoma (Kuang et al., 2005). A. flavus also causes mycoses (infection with fungus as opposed to diseases caused by consumption of fungal toxins which are broadly known as mycotoxicoses) in humans and animals. A. flavus is unique in that it is an opportunistic pathogen of both plants and animals (Gauthier and Keller, 2013; Hedayati et al., 2007). In humans, A. flavus is the second most common cause of invasive aspergillosis accounting for 10–20% of infections; only second to A. fumigatus which accounts for 80–90% of invasive aspergillosis infections (Krishnan et al., 2009). In hot and dry areas like Africa and the Middle East, A. flavus causes the majority of cases of fungal sinusitis, keratitis and cutaneous infections (Khairallah et al., 1992; Krishnan et al., 2009). Animals including rabbits, chickens and turkey are also highly susceptible to aspergillosis from A. flavus infection. In this review, we will highlight genes and molecules important in secondary metabolism and development of A. flavus and related species. The reader is also referred to other reviews for greater coverage of specific areas of research on this fungus and/or aflatoxin biosynthesis (Chang and Ehrlich, 2013; Khlangwiset et al., 2011; Woloshuk and Shim, 2013) as well as on environmental factors that influence host-pathogen interaction between A. flavus and maize (Fountain et al., 2013). 2. A. flavus genome: secondary metabolite gene clusters A. flavus belongs to Aspergillus section Flavi, which at latest assessment contains 22 species including the plant pathogen A. parasiticus and the industrial/food use Aspergilli A. oryzae and A. sojae (Varga et al., 2011). All Aspergilli have 8 chromosomes, however, A. flavus and the closely related A. oryzae have larger

genomes (37 Mb) compared to genomes of A. fumigatus (30 Mb), A. nidulans (31 Mb) and most other Aspergilli. The genome of A. flavus encodes 12,000 functional genes and contains extra copies of some lineage-specific genes giving rise to the larger genome (Rokas et al., 2007). The genome of A. flavus has been predicted to contain 56 secondary metabolite clusters (55 clusters identified by Georgianna et al., 2010 and the kojic acid cluster identified by Marui et al., 2011). However, using a program termed MIDDAS-M (motif-independent de novo detection algorithm for SMB gene clusters, where SMB refers to secondary metabolite biosynthesis), Umemura and colleagues identified several more secondary metabolite clusters using the same microarray data generated by Georgianna et al. (Umemura et al., 2013). It is therefore safe to say that the total number of clusters remains unknown; below we will describe the eight that have been fully or partially characterized. The aflatoxin cluster, which contains aflatoxin biosynthesis genes as well as pathway-specific regulatory genes, in total consists of 25 genes spanning a 70 kb DNA section (Yu et al., 2004a) and has been the subject of numerous reviews (Amaike et al., 2013a; Yu, 2012Yu et al., 2004b). The aflatoxin cluster is located close to the telomere of chromosome three and is flanked by four putative sugar-utilization genes (Yu et al., 2000) on the distal end and the cyclopiazonic acid cluster (Chang et al., 2009b) on the proximal end. Aflatoxin biosynthesis requires a complex regulatory mechanism orchestrated by the pathway-specific regulatory genes, aflR and to a lesser extent aflS (formerly aflJ). aflR encodes a DNA-binding, zinc-cluster protein that binds a palindromic sequence (50 TCGN5CGA-30 ) in the promoter region of aflatoxin pathway genes and activates their expression (Ehrlich et al., 1999; Fernandes et al., 1998; Yu et al., 1996, Table 1). AflR is an absolute requirement for the activation of most aflatoxin pathway genes as aflR deletion leads to complete loss of aflatoxin synthesis (Price et al., 2006; Woloshuk et al., 1994). Moreover, when aflR is overexpressed, increased transcript levels of aflatoxin pathway genes and aflatoxin are observed (Flaherty and Payne, 1997). However, some genes in the aflatoxin pathway may be only partly regulated by AflR as gene expression is present in aflR deletion mutants, albeit at lower levels than in wild type (Price et al., 2006). Both the aflatoxin pathway and AflR are conserved in the model fungus, A. nidulans, where the pathway terminates with production of the aflatoxin precursor, sterigmatocystin (Brown et al., 1996). Much of our knowledge of AflR regulation comes from this species (Fernandes et al., 1998). Aflatoxin biosynthesis is also regulated by aflS, another pathway-specific regulatory gene located divergently next to aflR. Separated by a small intergenic region, aflR and aflS have independent promoters but share joint regulation by transcription factors and other regulatory elements such as the bZIP protein RsmA (further discussed in Section 3). Unlike the well-defined role of AflR, that of AflS is still unclear because aflS deletion does not impact expression of aflatoxin pathway genes, however, A. flavus is still unable to make aflatoxin if this gene is deleted (Meyers et al., 1998). Potentially, AflS impacts aflatoxin biosynthesis through acting as a transcriptional enhancer or co-activator of AflR as it was shown to interact with AflR (Chang, 2003). Moreover, a synergistic relationship between AflR and AflS has also been reported in A. parasiticus where strains transformed with both aflR and aflS produced significantly more aflatoxin precursors than the strains transformed with just aflR (Chang et al., 2002). Most recently, a new study found that AflS was required for proper transport of AflR to or from the nucleus and thus may assist in localization rather than – or in addition to – activation processes (Ehrlich et al., 2012). Mutations in both aflR and aflS have been associated with atoxigenicity in the food fermentation fungi A. oryzae and A. sojae (Chang, 2004). aflR and aflS are conserved not only in Aspergilli but even also in the

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Table 1 A summary table of A. flavus, A. parasiticus or A. oryzae genes discussed in review and their impact on development and secondary metabolism. Y means deletion or overexpression of the gene impacts development of conidia, sclerotia or secondary metabolism. NA = data not available. Example references are listed under sources. Gene

Conidia production

Sclerotia production

Secondary metabolism

Sources

aflR aflS laeA

NA NA Y

Y Y Y

Y Y Y

veA velB vosA velC ppoA ppoB ppoC ppoD Aflox gprC gprD fluG nsdC nsdD meaB atfA atfB ApyapA msnA srrA Aflatoxin cluster Aflatrem cluster Cyclopiazonic acid cluster lna/lnb clusters PKS cluster Kojic acid cluster

Y Y Y N Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y NA Y NA NA

Y Y NA N Y Y Y Y Y Y Y Y Y Y NA NA NA NA NA NA Y NA NA

Y Y NA N Y Y Y Y Y Y Y NA Y Y Y Y Y Y Y Y Y Y Y

Chang et al. (2002) and Yu et al. (1996) Chang et al. (2002) and Meyers et al. (1998) Bayram et al. (2008), Bok and Keller (2004), and Sarikaya Bayram et al. (2010) Bayram et al. (2008) Bayram et al. (2008) Sarikaya Bayram et al. (2010) Chang et al. (2013) Brown et al. (2009) Brown et al. (2009) Brown et al. (2009) Brown et al. (2009) Brown et al. (2009) Affeldt et al. (2012) Affeldt et al. (2012) Chang et al. (2012a) Cary et al. (2012) Cary et al. (2012) Amaike et al. (2013b) Lara-Rojas et al. (2011) and Temme et al. (2012) Roze et al. (2011) and Sakamoto et al. (2008) Reverberi et al. (2008) Chang et al. (2011) Hong et al. (2013b) Chang et al. (2002), Kale et al. (1996), and Price et al. (2006) Nicholson et al. (2009) and Zhang et al. (2004) Chang et al. (2009b)

NA NA Y

Y NA NA

Y NA Y

Forseth et al. (2013) Cary et al. (2014) Marui et al. (2011)

pine tree pathogen, Dothistroma septosporum, which produces dothistromin, a mycotoxin analogous to aflatoxin and sterigmatocystin precursors. Dothistromin is unique in that its biosynthesis genes are not clustered but spread across a single chromosome (chromosome 12) yet they are regulated by AflR (Chettri et al., 2013). In addition to the aflatoxin cluster, A. flavus secondary metabolite clusters have also been partly characterized for the mycotoxins aflatrem and cyclopiazonic acid as well as the recently described piperazine molecules derived from two homologous NRPS-like gene clusters, a sclerotia-specific pigment, asparasone, produced from a polyketide gene cluster and ustiloxin B. Moreover, the kojic acid cluster has been identified in A. oryzae, a species considered a non-aflatoxigenic version of A. flavus. Aflatrem, an indole-diterpene, is a potent tremorgenic mycotoxin that causes neural disorders (Gallagher and Wilson, 1979). Unlike most secondary metabolite clusters whose genes are clustered at a single locus, the aflatrem biosynthesis genes are clustered at two different loci on two different chromosomes. The aflatrem cluster consists of eight genes, with three genes, atmG, atmC and atmM, located on one locus, ATM1, found on the telomere proximal on chromosome five. ATM2, the second locus located on chromosome seven, holds the other five genes, atmD, atmQ, atmB, atmA and atmP (Nicholson et al., 2009). The roles of some aflatrem biosynthesis genes were elucidated by functionally substituting them for paxilline biosynthesis genes in Penicillium paxilli, the model organism for studying indole-diterpenes. For example, it was shown that the aflatrem biosynthesis gene atmP could functionally substitute for the P. paxilli paxP gene and yield a P. paxilli strain able to produce paxilline, a secondary metabolite structurally similar to aflatrem (Nicholson et al., 2009). Moreover, an earlier study also showed that atmM has the capability to complement paxM mutants

of P. paxilli and successfully synthesize paxilline, indicating that atmM is a functional homolog of paxM (Zhang et al., 2004). Cyclopiazonic acid, a mycotoxin produced by various species of Aspergillus and Penicillium, is an indole-tetramic acid and a member of the family of indole-drived ergot alkaloids. Cyclopiazonic acid elicits its toxic effect through inhibition of calcium-dependent ATPase in the sarcoplasmic reticulum, causing calcium ion imbalance, which ultimately leads to increased muscular contractions (Goeger and Riley, 1989). Cyclopiazonic acid biosynthesis genes were speculated to be physically linked to the aflatoxin gene cluster because truncation of the aflatoxin cluster and its neighboring subtelomeric regions led to loss of aflatoxin as well as cyclopiazonic acid production. When three genes, including a monoamine oxidase, a dimethylallyl tryptophan synthase (required for cyclopiazonic acid synthesis), and a hybrid polyketide non-ribosomal peptide synthase, located in the subtelomeric region of an aflatoxigenic A. flavus strain were disrupted, cyclopiazonic acid production was abolished (Chang et al., 2009b). A recent review thoroughly addresses toxicity issues and biosynthesis of this mycotoxin (Chang et al., 2009a). Kojic acid, a skin-lightening cosmetic, is a secondary metabolite produced by some Aspergilli including A. flavus, A. parasiticus and A. oryzae as well as some Penicillium species (Parrish et al., 1966). Although kojic acid was first isolated in 1907, genes involved in its biosynthesis have only recently been characterized. Three genes, encoding for an enzyme (kojA), a transporter (kojT) and a transcription factor (kojR), were found to abolish kojic acid synthesis when deleted in A. oryzae. Similar to other secondary metabolism genes, kojA and kojT are closely associated in the A. oryzae genome at chromosome 5. kojR, located between kojA and kojT, is a Zn(II)2Cys6 transcriptional activator that is required for the activation of both kojA and kojT. Interestingly, kojic acid biosynthesis is

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regulated by a positive feedback-mechanism in which the product, kojic acid, induces activation of kojA and kojT in the presence of kojR. To coordinate the positive feedback loop, it is postulated that kojR is constitutively expressed at low levels, causing the accumulation of kojA and kojT transcripts, which lead to kojic acid synthesis. Kojic acid, initially produced at low levels, reaches a threshold and then highly induces kojA and kojT resulting in increased kojic acid biosynthesis (Marui et al., 2011; Terabayashi et al., 2010). Recently, several new gene clusters were characterized in A. flavus, two of which are the homologous gene clusters, lna and lnb, which contain highly homologous NRPS-like genes, lnaA and lnbA. Unlike canonical NRPSs that contain an adenylation domain, a peptidyl carrier protein, a thioester reductase domain and a condensation domain, LnaA and LnbA (58% identical at the amino acid level) lack a condensation domain. LnaA and LnbA produce redundant tyrosine-derived small-molecules and down regulation of both genes, but not either one individually, led to severe repression of sclerotia formation (Forseth et al., 2013). Another new cluster identified in A. flavus is the polyketide synthase gene cluster that produces a sclerotia-specific pigment, asparasone, under regulation by the Velvet Complex proteins, VeA and LaeA (Section 3). Deletion of the polyketide synthase in this cluster results in abnormal greyish-yellow sclerotia that were more susceptible to insect predation as well as damage by UV and heat (Cary et al., 2014). The MIDDASM program detected a new cluster responsible for production of ustiloxin B. The borders of this cluster have not been defined and it may contain between 13 and 18 genes (Umemura et al., 2013). The role of this compound in A. flavus biology has not been explored but it has been reported as a phytotoxin and mycotoxin produced by the fungus Ustilaginoidea virens (Koiso et al., 1992). 3. Global regulation by the Velvet Complex A conserved regulatory unit in dimorphic and filamentous fungi called the Velvet Complex is involved in regulating biosynthesis of multiple secondary metabolites (Table 2). The Velvet Complex is composed of the proteins VeA, LaeA and VelB, which form a heterotrimer in the nucleus to coordinate and control fungal development and secondary metabolism (Bayram et al., 2008). Moreover, Velvet Complex proteins also interact with other proteins to impact both development and secondary metabolism such as the nuclear interaction between VelB and VosA (a protein that confers spore viability and also regulates asexual development) resulting in repression of asexual development (Sarikaya Bayram et al.,

2010). Another velvet family member, VelC, did not impact either development or toxin production when deleted in A. flavus (Chang et al., 2013) and minimally affected conidiation when deleted in the fungus Fusarium oxysporum (Lopez-Berges et al., 2013). The Velvet Complex was first discovered in the model fungus Aspergillus nidulans but has since been described in numerous fungi including the filamentous fungal genera Fusarium, Cochliobolus, Penicillium, Trichoderma, Botrytis and Magnaporthe, the dimorphic fungus Histoplasma (reviewed in Jain and Keller, 2013) and most recently, at least some members in the basidiomycete Ustilago maydis (Karakkat et al., 2013). In A. flavus, studies of veA and laeA mutants show both genes are required for the production of aflatoxin (Amaike and Keller, 2009; Duran et al., 2007; Kale et al., 2008) and microarray data of deletions of both genes shows they are global in regulation of secondary metabolites (Cary et al., 2007; Georgianna et al., 2010). As also observed in other fungi, these proteins are essential for normal development in A. flavus. In A. flavus, both laeA and veA deletion lead to loss of sclerotia production as well as reduction in conidiation (Duran et al., 2007; Kale et al., 2008). Moreover, the deletion of both veA and laeA also appears to disrupt quorum sensing in A. flavus as will be discussed further in Section 4 (Amaike and Keller, 2009). On host seeds, veA and laeA deletion strains are characterized by reduced pathogenicity. Both DlaeA and DveA strains of A. flavus produced fewer conidia (DveA far less than DlaeA strains) and no aflatoxin on maize and peanut seeds. Furthermore, while the DlaeA strains could invade host cells intracellularly, hyphae of DveA strains could only grow intercellularly in epidermal cells. Both the DlaeA and DveA strains of this fungus had significantly impaired ability to degrade host cell lipid reserves when compared to wild type strains (Amaike and Keller, 2009). This may be related to an earlier study that showed lipase gene expression was correlated with pathogenicity (Yu et al., 2003). Recently, it was shown that laeA deletion in A. flavus impacts conidial hydrophobicity such that the DlaeA strains had more hydrophilic conidia (Chang et al., 2012b). This could potentially be linked to loss of aflatoxin production in DlaeA strains as the reduced hydrophobicity could impact formation of vesicles, which have been shown to be important in aflatoxin synthesis and transport in A. parasiticus (Chanda et al., 2009). Defects of laeA and veA mutants in secondary metabolism may be partially rescued by some proteins such as the bZIP protein RsmA, which in A. nidulans laeA and veA mutants restores sterigmatocystin biosynthesis through induction of aflR and aflS (Shaaban et al., 2010; Yin et al., 2012, 2013).

Table 2 Comparison of conserved pathways regulating development and secondary metabolism in three Aspergillus species. Yes = has an impact on specific process in the species indicated. NA = data not available. Example references are listed under sources. A. flavus

A. fumigatus

A. nidulans

Sources

Velvet Complex Secondary metabolism

Yes

Yes

Yes

Asexual sporulation

Yes

Yes

Yes

Sexual development

Yes

NA

Yes

Bayram et al. (2008), Bok et al. (2005), Bok and Keller (2004), Kale et al. (2008), Park et al. (2012), Perrin et al. (2007), and Sarikaya Bayram et al. (2010) Bayram et al. (2008), Bok et al. (2005), Kale et al. (2008), Park et al. (2012), Sarikaya Bayram et al. (2010) Bayram et al. (2008), Kale et al. (2008), Sarikaya Bayram et al. (2010)

Stress response network Secondary metabolism

Yes

NA

Yes

Asexual sporulation Sexual development

Yes Yes

NA NA

Yes Yes

Chang et al. (2011), Hong et al. (2013b), Reverberi et al. (2007, 2008), Roze et al. (2011), Temme et al. (2012), and Yin et al. (2013) Chang et al. (2011) and Yin et al. (2013) Chang et al. (2011) and Yin et al. (2013)

Ppo oxygenases Secondary metabolism Asexual sporulation Sexual development Quorum signaling

Yes Yes Yes Yes

NA Yes NA NA

Yes Yes Yes Yes

Brown et al. (2009) and Tsitsigiannis and Keller (2006) Dagenais et al. (2008), Brown et al. (2009), and Tsitsigiannis and Keller (2006) Brown et al. (2009) and Tsitsigiannis and Keller (2006) Affeldt et al. (2012) and Herrero-Garcia et al. (2011)

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4. Quorum sensing in A. flavus Filamentous fungi follow a programmed developmental sequence from spore germination to vegetative hyphae, which can differentiate into either sexual or asexual sporulation structures. Accumulating evidence has shown that fungi regulate development, and more recently secondary metabolism, through quorum sensing, a density-dependent phenomenon that leads to a coordinated population-wide response (Albuquerque and Casadevall, 2012). Most studies have focused on yeast and the basidiomycete Cryptococcus neoformans with signaling molecules ranging from small alcohols to various fatty acids. A. flavus reproduces in a density-dependent manner where low population densities are characterized by increased sclerotia production and reduced conidiation (Brown et al., 2009; Horowitz Brown et al., 2008). As the population transitions to high cell density, the reverse phenotype is observed with reduced sclerotia production and increased conidiation. Moreover, A. flavus also responds to spent-medium extracts producing more conidia or sclerotia when exposed to high- or low-density spent-medium extracts respectively. In addition to development, secondary metabolism is also regulated in a cell density-dependent manner where aflatoxin biosynthesis pattern mirrors that of sclerotia production; much higher production at low population density (Affeldt et al., 2012; Horowitz Brown et al., 2008). Several molecules and genes have been found to be important in quorum sensing in A. flavus. Chief among these are oxylipins, a group of diverse oxygenated polyunsaturated fatty acids that act as molecular signals across the fungal, plant and animal kingdoms. Collectively referred to as psi (precocious sexual inducer) factors in Aspergillus species, oxylipins are known to regulate the balance between asexual and sexual development in Aspergillus species including A. flavus (Calvo et al., 1999, 2001). In A. nidulans, when oxylipin-encoding dioxygenase genes (ppo genes) were deleted, the balance between sexual and asexual development was disrupted (Tsitsigiannis and Keller, 2007). Similarly, simultaneous disruption of A. flavus ppo genes (ppoA, ppoB, ppoC and ppoD) together with another oxylipin-generating gene, lox, caused disruption in the normal morphological development and abolished the switch from sclerotia to conidia even as population density transitioned from low to high density. Moreover, the ppoC and lox deletion mutant strains also consistently produced high levels of aflatoxin at any population density (Horowitz Brown et al., 2008; Brown et al., 2009). VeA and LaeA have also been found to be important in quorum sensing as deletion of either gene results in failure to produce sclerotia at any density (Amaike and Keller, 2009). Oxylipins also possibly play a role in pathogenicity, as loss of several of the oxylipin genes in A. flavus has been associated with altered pathogenicity on host seeds. In the study discussed above, when all four ppo genes and the lox gene were disrupted simultaneously in A. flavus, the mutant strains showed markedly reduced conidiation but increased aflatoxin production on maize and peanut seeds (Brown et al., 2009). In another study, A. flavus ppoA, ppoB and ppoC were expressed during pathogenesis on hazelnut with ppoB appearing to be expressed exclusively during pathogenesis (Gallo et al., 2010). Oxylipins may also potentially be involved in fungus-host cross-communication as plant-derived oxylipins (9(S)-HpODE) can functionally substitute for fungal-derived oxylipins and stimulate sporulation (Brodhagen et al., 2008; Calvo et al., 1999). Oxylipins have also been implicated in injury-response mechanisms that may underlie some of the developmental response in fungi (Hernandez-Onate et al., 2012) as well as ability of fungal spores to germinate (Herrero-Garcia et al., 2011). Until recently, how fungi perceive oxylipins was unknown, but there is accumulating evidence that oxylipins are sensed by

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G protein-coupled receptors (GPCRs). GPCR deletion mutants in A. flavus and other filamentous fungi exhibit disruption in development and secondary metabolism. A recent study investigating two A. flavus GPCRs, GprC and GprD, found that their deletion led to impaired ability to transition from low to high cell-density phenotypes as both deletion strains produced high sclerotia and aflatoxin levels even in unfavorable high population density conditions (Affeldt et al., 2012). 5. Conserved Aspergillus proteins involved in morphogenesis in A. flavus In addition to the Velvet Complex proteins and oxygenases discussed above, various other proteins are also known to regulate fungal development and/or secondary metabolism in A. flavus. nsd (never in sexual development) genes, initially discovered from A. nidulans mutants that failed to produce cleistothecia (Han et al., 1998), control development and secondary metabolism in A. nidulans (Han et al., 2001). Two nsd genes, both encoding for GATA-type transcription factors, act similarly in A. flavus with nsdC and nsdD deletion mutants showing reduced conidiation (arising from aberrant conidiophore morphology), loss of sclerotia formation and loss of aflatoxin production (Cary et al., 2012). Another gene, fluG, which in A. nidulans is required for conidiation and sterigmatocystin production, was found to affect development but not secondary metabolism in A. flavus. A. nidulans fluG mutants are completely impaired in conidia production (Lee and Adams, 1994) because the mutants are unable to produce a diffusible factor required for sporulation that was recently characterized to be an adduct of the meroterpenoids dehydroaustinol and diorcinol (Rodriguez-Urra et al., 2012). A. nidulans fluG deletion mutants are also unable to biosynthesize sterigmatocystin (Hicks et al., 1997). In contrast, in addition to highly increased sclerotia production, A. flavus fluG mutants only exhibit a delay and reduction in conidia production and no impact on aflatoxin production (Chang et al., 2012a). FluG potentially modulates sclerotia production through interaction with VelB as the two proteins have been shown to interact. Moreover, fluG deletion in DvelB strains further significantly decreases conidiation even in conidiation-inducing light environment (Chang et al., 2013). FluG interacts with a conserved G protein signaling system linking asexual sporulation with secondary metabolism. Briefly, FluG activates a heterotrimeric G protein complex that ultimately signals through protein kinase A (PkaA) to activate AflR (Shimizu et al., 2003). Although this system was first worked out in A. nidulans (reviewed in Brodhagen and Keller, 2006; Park and Yu, 2012), several of the orthologs have been found to be conserved in A. parasiticus (and thus likely in A. flavus) (Hicks et al., 1997; Roze et al., 2004). 6. Oxidative stress response in secondary metabolism and development Oxidative stress response and secondary metabolism are thought to be highly integrated processes. Many filamentous fungi, including A. flavus, A. parasiticus, A. oryzae and A. nidulans, exhibit a close association and interplay between these two processes where secondary metabolism is often induced as a response to cellular oxidative stress (for more in-depth reviews on the linkage between oxidative stress response and secondary metabolism and development, the reader is referred to Hong et al., 2013a; Montibus et al., 2013). Various proteins, many belonging to the bZIP transcription factor family, coordinate this interplay between oxidative stress and secondary metabolism. bZIP proteins such as AtfB, AtfA and AP-1 (and its orthologs ApYapA and NapA) play key roles in

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co-regulation of these two processes in many filamentous fungi. In A. parasiticus, not only does atfB expression correlate with aflatoxin production, but AtfB also binds to promoters of aflatoxin biosynthesis genes as well as stress response genes (Hong et al., 2013b; Roze et al., 2011). Moreover, accumulation of aflatoxin biosynthesis gene transcripts as well as accumulation of atfB transcript occurs concurrently during aflatoxin production (Roze et al., 2011). In further evidence of the co-regulation of oxidative stress and secondary metabolism, AtfB and another oxidative stress response-related bZIP protein, AP-1 (an ortholog of ApYapA), heterodimerize to promote transcription of nor-1, an aflatoxin biosynthesis gene (Roze et al., 2011). Furthermore, when ApyapA was deleted in A. parasiticus, the mutants were characterized by increased susceptibility to extracellular oxidants. The mutants also exhibited increased formation of precocious reactive oxygen species and increased aflatoxin production (Reverberi et al., 2008). In an earlier related study, DApyap1 strains of A. parasiticus exhibited increased pathogenicity on maize seeds with earlier and more production of aflatoxins (Reverberi et al., 2007). In A. nidulans, when the ApyapA homolog, napA, was overexpressed, mutants showed more robust resistance to oxidative stress and decreased secondary metabolism synthesis (Yin et al., 2013). To a lesser extent, another bZIP protein, AtfA, potentially also co-regulates oxidative stress and secondary metabolism. In A. nidulans, AtfA is known to regulate oxidative stress response (Balázs et al., 2010), while an A. nidulans AtfA ortholog in the plant pathogen Botrytis cinerea (BcAtf1) regulates secondary metabolism as its deletion (Dbcatf1) leads to much higher accumulation of B. cinerea secondary metabolites (Temme et al., 2012). Aside from bZIP proteins, other proteins such as MsnA and SrrA also co-regulate oxidative stress response and secondary metabolism. MsnA (ortholog of multi-stress response S. cerevisiae Msn2 protein) is a zinc-finger, stress-related protein that when deleted (DmsnA) leads to increased biosynthesis of secondary metabolites aflatoxin and kojic acid in both A. parasiticus and A. flavus. Moreover, the DmsnA strains of both A. flavus and A. parasiticus showed increased production of reactive oxygen species (Chang et al., 2011). Recently, a novel conserved motif in the promoter regions of both aflatoxin biosynthesis and oxidative stress response genes was discovered and the transcription factor protein SrrA (an ortholog of the oxidative stress response-related yeast Skn7 protein) was found to bind to this novel motif (Hong et al., 2013b). In filamentous fungi, bZIP proteins play ubiquitous roles in fungal life; they coordinate oxidative stress response, secondary metabolism as well as development. The bZIP proteins discussed above, AtfA, AtfB and ApYapA and its homolog NapA, are known to confer reactive oxygen species tolerance to conidia and/or hyphae (Hong et al., 2013a; Montibus et al., 2013; Sakamoto et al., 2008). While not examined for oxidative stress responses, other bZIP proteins are important in Aspergillus biology. In A. fumigatus, the flbB gene encodes for two bZIP proteins, AfuFlbBa and AfuFlbBb, which are required for normal conidiation and secondary metabolite production (Xiao et al., 2010). Another bZIP protein, the nitrogen-regulatory MeaB protein, is important in regulating virulence and secondary metabolism. When overexpressed, OE::meaB A. flavus strains exhibited phenotypes of decreased host seed colonization, reduced lipase activity and loss of aflatoxin synthesis mirroring the phenotype of DlaeA strains (Amaike et al., 2013b). Also, as noted in Section 3, RsmA is important in sterigmatocystin synthesis in A. nidulans.

7. Conclusion Over 50 years of intense research has revealed much about the genes, molecules and factors that control the intricate process of

aflatoxin biosynthesis in A. flavus, with newer studies revealing pathways and molecules important for development, pathogenesis and secondary metabolism. The current data supports a multifactorial complex underlying virulence which involves production of many secondary metabolites – not just aflatoxin, Velvet Complex members VeA and LaeA, a G protein-PkaA signaling pathway, several stress response systems and a quorum sensing mechanism. It is likely that our molecular understanding of this pathogen will accelerate over the next few years due to the availability of fungal genome sequence that has allowed for coverage of gene expression over various growth conditions including pathogenesis, life cycle progression and stress responses leading to discovery of secondary metabolites and other molecules important to fungal biology (Brakhage and Schroeckh, 2011; Chiang et al., 2008, 2009; Kale et al., 2008; Lim et al., 2012; Martens-Uzunova and Schaap, 2009; Schneider et al., 2007; Wang et al., 2010). While in-depth coverage of all of the microarray and RNAseq studies is beyond the goal of this review; the reader is directed to specific papers which have added valuable information on genes and pathways contributing to A. flavus virulence and development (Cary et al., 2007; Georgianna et al., 2010; Gibbons et al., 2012; Lin et al., 2013; Olarte et al., 2012; Reese et al., 2011; Yu et al., 2011). We hope that analysis of these studies will lead to discovery of genes and molecules involved in pathogenicity or fungal survival that may contribute to efforts to control diseases caused by this unique species, not just as a plant pathogen but also as its role as an opportunistic human pathogen. Acknowledgment We thank Katharyn J. Affeldt for critical commentary on this review. References Affeldt, K.J. et al., 2012. Aspergillus oxylipin signaling and quorum sensing pathways depend on G protein-coupled receptors. Toxins (Basel) 4, 695–717. Albuquerque, P., Casadevall, A., 2012. Quorum sensing in fungi – a review. Med. Mycol. 50, 337–345. Amaike, S. et al., 2013a. Genetics, biosynthesis and regulation of aflatoxins and other Aspergillus flavus secondary metabolites. In: Kempken, F. (Ed.), Agricultural Applications. Springer, Berlin, Heidelberg, pp. 59–74. Amaike, S. et al., 2013b. The bZIP protein MeaB mediates virulence attributes in Aspergillus flavus. PLoS One 8, e74030. Amaike, S., Keller, N.P., 2011. Aspergillus flavus. Annu. Rev. Phytopathol. 49, 107– 133. Amaike, S., Keller, N.P., 2009. Distinct roles for VeA and LaeA in development and pathogenesis of Aspergillus flavus. Eukaryot. Cell 8, 1051–1060. Azziz-Baumgartner, E. et al., 2005. Case-control study of an acute aflatoxicosis outbreak, Kenya, 2004. Environ. Health Perspect. 113, 1779–1783. Balázs, A. et al., 2010. AtfA bZIP-type transcription factor regulates oxidative and osmotic stress responses in Aspergillus nidulans. Mol. Genet. Genom. 283, 289– 303. Bayram, O. et al., 2008. VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320, 1504–1506. Bok, J.W. et al., 2005. LaeA, a regulator of morphogenetic fungal virulence factors. Eukaryot. Cell 4, 1574–1582. Bok, J.W., Keller, N.P., 2004. LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryot. Cell 3, 527–535. Brakhage, A.A., Schroeckh, V., 2011. Fungal secondary metabolites – strategies to activate silent gene clusters. Fungal Genet. Biol. 48, 15–22. Brodhagen, M., Keller, N.P., 2006. Signalling pathways connecting mycotoxin production and sporulation. Mol. Plant Pathol. 7, 285–301. Brodhagen, M. et al., 2008. Reciprocal oxylipin-mediated cross-talk in the Aspergillus-seed pathosystem. Mol. Microbiol. 67, 378–391. Brown, D.W. et al., 1996. Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans. Proc. Natl. Acad. Sci. USA 93, 1418–1422. Brown, S.H. et al., 2009. Oxygenase coordination is required for morphological transition and the host-fungus interaction of Aspergillus flavus. Mol. Plant Microbe Interact. 22, 882–894. Calvo, A.M. et al., 2001. Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans. J. Biol. Chem. 276, 25766–25774. Calvo, A.M. et al., 1999. Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp. Appl. Environ. Microbiol. 65, 3668–3673.

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