Fungal Genetics and Biology 66 (2014) 11–18

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Fungal Genetics and Biology

journal homepage: www.elsevier .com/locate /yfgbi

Review

Molecular mechanisms of Aspergillus flavus secondary metabolismand development

http://dx.doi.org/10.1016/j.fgb.2014.02.0081087-1845/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author.E-mail address: npkeller@wisc.edu (N.P. Keller).

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 i n f o

Article history:Received 3 January 2014Accepted 25 February 2014Available online 5 March 2014

Keywords:Aspergillus flavusLaeAVeAAflatoxinGene clusterQuorum

a b s t r a c t

The plant and human opportunistic fungus Aspergillus flavus is recognized for the production of thecarcinogen aflatoxin. Although many reviews focus on the wealth of information known about aflatoxinbiosynthesis, few articles describe other genes and molecules important for A. flavus development orsecondary 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 signalingpathways) and the function of the transcriptional regulatory unit known as the Velvet Complex. Acomparison to other Aspergilli reveals conservation in several pathways affecting fungal developmentand metabolism.

� 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112. A. flavus genome: secondary metabolite gene clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123. Global regulation by the Velvet Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144. Quorum sensing in A. flavus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155. Conserved Aspergillus proteins involved in morphogenesis in A. flavus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156. Oxidative stress response in secondary metabolism and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1. Introduction

Aspergillus flavus is a ubiquitous saprophytic fungus found insoils across the world. Although first described in 1809, A. flavuswas thrown into the limelight in 1962 as a result of the Turkey Xdisease that killed thousands of poultry (Nesbitt et al., 1962). TheTurkey X outbreak led to the discovery of aflatoxin, a fungal myco-toxin that had contaminated the poultry feed. Since then, A. flavusand aflatoxin have had tremendous economic and health impactsacross the world (Amaike and Keller, 2011). Although aflatoxin isnoted as the primary metabolite causing human disease, the

fungus produces several toxic metabolites that may also contributeto ill health as covered below.

A. flavus survives as conidia or sclerotia in soil and organic deb-ris. While conidia allow the fungus to mass-disseminate, sclerotiaenable survival in harsh environmental conditions and can germi-nate once conditions improve. On host tissue, including that of hu-mans, animals and plants, conidia germinate and grow as myceliawhich can develop into either conidiophores or sclerotia depend-ing on environmental and nutritional cues. While the conidium isconsidered the predominant infectious spore, the predominantreproductive form in soil is not known. Sclerotia contain the sexualascospores of the fungus, which until recently were only reportedto occur in experimental laboratory conditions but have now beenreported to occur in the field also (Horn et al., 2009, 2013).

12 M.G. Amare, N.P. Keller / Fungal Genetics and Biology 66 (2014) 11–18

A. flavus colonizes and produces aflatoxins (there are severalforms of aflatoxin with aflatoxin B1 being the most carcinogenic)in oil-rich agricultural crops including maize, peanuts and cotton-seeds both pre- and post-harvest. As aflatoxin is toxigenic as wellas carcinogenic, controlling aflatoxin contamination of crops isvital. However, controlling contamination, both pre- and post-harvest, induces tremendous monetary losses worldwide. Inthe US, aflatoxin contamination incurs economic losses ofapproximately US $1 billion per annum (Vardon et al., 2003) whileAfrican countries lose approximately $670 million from failure tomeet European export standards (Otsuki et al., 2001). In an effortto curb aflatoxin exposure, the US Food and Drug Administrationonly allows 20 ppb in food and 0.5 ppb in milk (Georgianna andPayne, 2009) while some countries such as those in Europe haveeven stricter guidelines. On the other hand, developing countriesoften have lax, if any, guidelines for aflatoxin contamination.

Aflatoxins have a wide range of health impacts depending onthe aflatoxin dose. Acute aflatoxicosis, arising from high-dose afla-toxin intake over a short period, often results in aflatoxin-poisoning outbreaks killing scores of people. The quintessentialexamples for this are the recurrent outbreaks seen in the EastAfrican country Kenya, which experienced its worst outbreak in2004 with 317 cases and 125 reported deaths (Azziz-Baumgartneret al., 2005). Chronic aflatoxicosis, arising from low-dose aflatoxinconsumption over an extended period, can result in immune sup-pression, stunting and liver cancer. Aflatoxin-induced liver canceris known to arise from a mutation in the tumor suppressor gene,p53, in the liver (Hsu et al., 1991). Perhaps exacerbating theproblem is the fact that exposure to aflatoxin B1 in hepatitis Bvirus-endemic areas highly increases the chances of developinghepatocellular carcinoma by as much as 30-fold, creating severehealth problems in developing countries where both are commonoccurrences (Groopman et al., 2008). To a lesser extent, aflatoxinB1 and hepatitis C virus also exhibit a similar relationship leadingto increased chances of developing hepatocellular carcinoma(Kuang et al., 2005).

A. flavus also causes mycoses (infection with fungus as opposedto diseases caused by consumption of fungal toxins which arebroadly known as mycotoxicoses) in humans and animals. A. flavusis unique in that it is an opportunistic pathogen of both plants andanimals (Gauthier and Keller, 2013; Hedayati et al., 2007). Inhumans, A. flavus is the second most common cause of invasiveaspergillosis accounting for 10–20% of infections; only second toA. fumigatus which accounts for 80–90% of invasive aspergillosisinfections (Krishnan et al., 2009). In hot and dry areas like Africaand the Middle East, A. flavus causes the majority of cases of fungalsinusitis, keratitis and cutaneous infections (Khairallah et al., 1992;Krishnan et al., 2009). Animals including rabbits, chickens andturkey are also highly susceptible to aspergillosis from A. flavusinfection.

In this review, we will highlight genes and molecules importantin secondary metabolism and development of A. flavus and relatedspecies. The reader is also referred to other reviews for greater cov-erage of specific areas of research on this fungus and/or aflatoxinbiosynthesis (Chang and Ehrlich, 2013; Khlangwiset et al., 2011;Woloshuk and Shim, 2013) as well as on environmental factorsthat influence host-pathogen interaction between A. flavus andmaize (Fountain et al., 2013).

2. A. flavus genome: secondary metabolite gene clusters

A. flavus belongs to Aspergillus section Flavi, which at latestassessment contains 22 species including the plant pathogenA. parasiticus and the industrial/food use Aspergilli A. oryzae andA. 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 ofA. flavus encodes 12,000 functional genes and contains extra copiesof 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 sec-ondary metabolite clusters (55 clusters identified by Georgiannaet al., 2010 and the kojic acid cluster identified by Marui et al.,2011). However, using a program termed MIDDAS-M (motif-inde-pendent de novo detection algorithm for SMB gene clusters, whereSMB refers to secondary metabolite biosynthesis), Umemura andcolleagues identified several more secondary metabolite clustersusing the same microarray data generated by Georgianna et al.(Umemura et al., 2013). It is therefore safe to say that the totalnumber of clusters remains unknown; below we will describethe eight that have been fully or partially characterized. The afla-toxin cluster, which contains aflatoxin biosynthesis genes as wellas pathway-specific regulatory genes, in total consists of 25 genesspanning a 70 kb DNA section (Yu et al., 2004a) and has been thesubject of numerous reviews (Amaike et al., 2013a; Yu, 2012Yuet al., 2004b). The aflatoxin cluster is located close to the telomereof chromosome three and is flanked by four putative sugar-utiliza-tion genes (Yu et al., 2000) on the distal end and the cyclopiazonicacid cluster (Chang et al., 2009b) on the proximal end.

Aflatoxin biosynthesis requires a complex regulatory mecha-nism orchestrated by the pathway-specific regulatory genes, aflRand to a lesser extent aflS (formerly aflJ). aflR encodes a DNA-bind-ing, zinc-cluster protein that binds a palindromic sequence (50-TCGN5CGA-30) in the promoter region of aflatoxin pathway genesand activates their expression (Ehrlich et al., 1999; Fernandeset al., 1998; Yu et al., 1996, Table 1). AflR is an absolute require-ment for the activation of most aflatoxin pathway genes as aflRdeletion leads to complete loss of aflatoxin synthesis (Price et al.,2006; Woloshuk et al., 1994). Moreover, when aflR is overexpres-sed, increased transcript levels of aflatoxin pathway genes andaflatoxin are observed (Flaherty and Payne, 1997). However, somegenes in the aflatoxin pathway may be only partly regulated byAflR as gene expression is present in aflR deletion mutants, albeitat lower levels than in wild type (Price et al., 2006). Both the afla-toxin pathway and AflR are conserved in the model fungus, A. nidu-lans, where the pathway terminates with production of theaflatoxin precursor, sterigmatocystin (Brown et al., 1996). Muchof our knowledge of AflR regulation comes from this species (Fer-nandes et al., 1998).

Aflatoxin biosynthesis is also regulated by aflS, another path-way-specific regulatory gene located divergently next to aflR. Sep-arated by a small intergenic region, aflR and aflS have independentpromoters but share joint regulation by transcription factors andother regulatory elements such as the bZIP protein RsmA (furtherdiscussed in Section 3). Unlike the well-defined role of AflR, thatof AflS is still unclear because aflS deletion does not impact expres-sion of aflatoxin pathway genes, however, A. flavus is still unable tomake aflatoxin if this gene is deleted (Meyers et al., 1998). Poten-tially, AflS impacts aflatoxin biosynthesis through acting as a tran-scriptional enhancer or co-activator of AflR as it was shown tointeract with AflR (Chang, 2003). Moreover, a synergistic relation-ship between AflR and AflS has also been reported in A. parasiticuswhere strains transformed with both aflR and aflS produced signif-icantly more aflatoxin precursors than the strains transformedwith just aflR (Chang et al., 2002). Most recently, a new study foundthat AflS was required for proper transport of AflR to or from thenucleus and thus may assist in localization rather than – or in addi-tion to – activation processes (Ehrlich et al., 2012). Mutations inboth aflR and aflS have been associated with atoxigenicity in thefood fermentation fungi A. oryzae and A. sojae (Chang, 2004). aflRand aflS are conserved not only in Aspergilli but even also in the

Table 1A 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 overexpressionof the gene impacts development of conidia, sclerotia or secondary metabolism. NA = data not available. Example references are listed under sources.

Gene Conidiaproduction

Sclerotiaproduction

Secondarymetabolism

Sources

aflR NA Y Y Chang et al. (2002) and Yu et al. (1996)aflS NA Y Y Chang et al. (2002) and Meyers et al. (1998)laeA Y Y Y Bayram et al. (2008), Bok and Keller (2004), and Sarikaya Bayram et al.

(2010)veA Y Y Y Bayram et al. (2008)velB Y Y Y Bayram et al. (2008)vosA Y NA NA Sarikaya Bayram et al. (2010)velC N N N Chang et al. (2013)ppoA Y Y Y Brown et al. (2009)ppoB Y Y Y Brown et al. (2009)ppoC Y Y Y Brown et al. (2009)ppoD Y Y Y Brown et al. (2009)Aflox Y Y Y Brown et al. (2009)gprC Y Y Y Affeldt et al. (2012)gprD Y Y Y Affeldt et al. (2012)fluG Y Y NA Chang et al. (2012a)nsdC Y Y Y Cary et al. (2012)nsdD Y Y Y Cary et al. (2012)meaB Y NA Y Amaike et al. (2013b)atfA Y NA Y Lara-Rojas et al. (2011) and Temme et al. (2012)atfB Y NA Y Roze et al. (2011) and Sakamoto et al. (2008)ApyapA Y NA Y Reverberi et al. (2008)msnA Y NA Y Chang et al. (2011)srrA NA NA Y Hong et al. (2013b)Aflatoxin cluster Y Y Y Chang et al. (2002), Kale et al. (1996), and Price et al. (2006)Aflatrem cluster NA NA Y Nicholson et al. (2009) and Zhang et al. (2004)Cyclopiazonic acid

clusterNA NA Y Chang et al. (2009b)

lna/lnb clusters NA Y Y Forseth et al. (2013)PKS cluster NA NA NA Cary et al. (2014)Kojic acid cluster Y NA Y Marui et al. (2011)

M.G. Amare, N.P. Keller / Fungal Genetics and Biology 66 (2014) 11–18 13

pine tree pathogen, Dothistroma septosporum, which producesdothistromin, a mycotoxin analogous to aflatoxin and sterigmato-cystin precursors. Dothistromin is unique in that its biosynthesisgenes 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 metabo-lite clusters have also been partly characterized for the mycotoxinsaflatrem and cyclopiazonic acid as well as the recently describedpiperazine molecules derived from two homologous NRPS-likegene clusters, a sclerotia-specific pigment, asparasone, producedfrom a polyketide gene cluster and ustiloxin B. Moreover, the kojicacid cluster has been identified in A. oryzae, a species considered anon-aflatoxigenic version of A. flavus.

Aflatrem, an indole-diterpene, is a potent tremorgenic myco-toxin that causes neural disorders (Gallagher and Wilson, 1979).Unlike most secondary metabolite clusters whose genes areclustered at a single locus, the aflatrem biosynthesis genes are clus-tered at two different loci on two different chromosomes. The afla-trem cluster consists of eight genes, with three genes, atmG, atmCand atmM, located on one locus, ATM1, found on the telomere prox-imal on chromosome five. ATM2, the second locus located on chro-mosome seven, holds the other five genes, atmD, atmQ, atmB, atmAand atmP (Nicholson et al., 2009). The roles of some aflatrem bio-synthesis genes were elucidated by functionally substituting themfor paxilline biosynthesis genes in Penicillium paxilli, the modelorganism for studying indole-diterpenes. For example, it wasshown that the aflatrem biosynthesis gene atmP could functionallysubstitute for the P. paxilli paxP gene and yield a P. paxilli strain ableto produce paxilline, a secondary metabolite structurally similar toaflatrem (Nicholson et al., 2009). Moreover, an earlier study alsoshowed that atmM has the capability to complement paxM mutants

of P. paxilli and successfully synthesize paxilline, indicating thatatmM is a functional homolog of paxM (Zhang et al., 2004).

Cyclopiazonic acid, a mycotoxin produced by various species ofAspergillus and Penicillium, is an indole-tetramic acid and a memberof the family of indole-drived ergot alkaloids. Cyclopiazonic acidelicits its toxic effect through inhibition of calcium-dependentATPase in the sarcoplasmic reticulum, causing calcium ion imbal-ance, which ultimately leads to increased muscular contractions(Goeger and Riley, 1989). Cyclopiazonic acid biosynthesis geneswere speculated to be physically linked to the aflatoxin gene clus-ter because truncation of the aflatoxin cluster and its neighboringsubtelomeric regions led to loss of aflatoxin as well as cyclopiazon-ic acid production. When three genes, including a monoamine oxi-dase, a dimethylallyl tryptophan synthase (required forcyclopiazonic acid synthesis), and a hybrid polyketide non-ribo-somal peptide synthase, located in the subtelomeric region of anaflatoxigenic A. flavus strain were disrupted, cyclopiazonic acidproduction was abolished (Chang et al., 2009b). A recent reviewthoroughly addresses toxicity issues and biosynthesis of this myco-toxin (Chang et al., 2009a).

Kojic acid, a skin-lightening cosmetic, is a secondary metaboliteproduced 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 inits biosynthesis have only recently been characterized. Threegenes, encoding for an enzyme (kojA), a transporter (kojT) and atranscription factor (kojR), were found to abolish kojic acid synthe-sis when deleted in A. oryzae. Similar to other secondary metabo-lism genes, kojA and kojT are closely associated in the A. oryzaegenome at chromosome 5. kojR, located between kojA and kojT, isa Zn(II)2Cys6 transcriptional activator that is required for the acti-vation of both kojA and kojT. Interestingly, kojic acid biosynthesis is

14 M.G. Amare, N.P. Keller / Fungal Genetics and Biology 66 (2014) 11–18

regulated by a positive feedback-mechanism in which the product,kojic acid, induces activation of kojA and kojT in the presence ofkojR. To coordinate the positive feedback loop, it is postulated thatkojR is constitutively expressed at low levels, causing the accumu-lation of kojA and kojT transcripts, which lead to kojic acid synthe-sis. Kojic acid, initially produced at low levels, reaches a thresholdand then highly induces kojA and kojT resulting in increased kojicacid biosynthesis (Marui et al., 2011; Terabayashi et al., 2010).

Recently, several new gene clusters were characterized inA. flavus, two of which are the homologous gene clusters, lna andlnb, which contain highly homologous NRPS-like genes, lnaA andlnbA. Unlike canonical NRPSs that contain an adenylation domain,a peptidyl carrier protein, a thioester reductase domain and a con-densation domain, LnaA and LnbA (58% identical at the amino acidlevel) lack a condensation domain. LnaA and LnbA produce redun-dant tyrosine-derived small-molecules and down regulation ofboth genes, but not either one individually, led to severe repressionof sclerotia formation (Forseth et al., 2013). Another new clusteridentified in A. flavus is the polyketide synthase gene cluster thatproduces a sclerotia-specific pigment, asparasone, under regulationby the Velvet Complex proteins, VeA and LaeA (Section 3). Deletionof the polyketide synthase in this cluster results in abnormal grey-ish-yellow sclerotia that were more susceptible to insect predationas well as damage by UV and heat (Cary et al., 2014). The MIDDAS-M program detected a new cluster responsible for production ofustiloxin B. The borders of this cluster have not been defined andit may contain between 13 and 18 genes (Umemura et al., 2013).The role of this compound in A. flavus biology has not been exploredbut it has been reported as a phytotoxin and mycotoxin producedby the fungus Ustilaginoidea virens (Koiso et al., 1992).

3. Global regulation by the Velvet Complex

A conserved regulatory unit in dimorphic and filamentous fungicalled the Velvet Complex is involved in regulating biosynthesis ofmultiple secondary metabolites (Table 2). The Velvet Complex iscomposed of the proteins VeA, LaeA and VelB, which form a hetero-trimer in the nucleus to coordinate and control fungal develop-ment and secondary metabolism (Bayram et al., 2008). Moreover,Velvet Complex proteins also interact with other proteins to im-pact both development and secondary metabolism such as the nu-clear interaction between VelB and VosA (a protein that confersspore viability and also regulates asexual development) resultingin repression of asexual development (Sarikaya Bayram et al.,

Table 2Comparison of conserved pathways regulating development and secondary metabolismindicated. NA = data not available. Example references are listed under sources.

A. flavus A. fumigatus A. nidulans

Velvet ComplexSecondary metabolism Yes Yes Yes

Asexual sporulation Yes Yes Yes

Sexual development Yes NA Yes

Stress response networkSecondary metabolism Yes NA Yes

Asexual sporulation Yes NA YesSexual development Yes NA Yes

Ppo oxygenasesSecondary metabolism Yes NA YesAsexual sporulation Yes Yes YesSexual development Yes NA YesQuorum signaling Yes NA Yes

2010). Another velvet family member, VelC, did not impact eitherdevelopment or toxin production when deleted in A. flavus (Changet al., 2013) and minimally affected conidiation when deleted inthe fungus Fusarium oxysporum (Lopez-Berges et al., 2013). TheVelvet Complex was first discovered in the model fungus Aspergil-lus nidulans but has since been described in numerous fungiincluding the filamentous fungal genera Fusarium, Cochliobolus,Penicillium, Trichoderma, Botrytis and Magnaporthe, the dimorphicfungus Histoplasma (reviewed in Jain and Keller, 2013) and mostrecently, at least some members in the basidiomycete Ustilagomaydis (Karakkat et al., 2013).

In A. flavus, studies of veA and laeA mutants show both genes arerequired for the production of aflatoxin (Amaike and Keller, 2009;Duran et al., 2007; Kale et al., 2008) and microarray data of dele-tions of both genes shows they are global in regulation of secondarymetabolites (Cary et al., 2007; Georgianna et al., 2010). As also ob-served in other fungi, these proteins are essential for normal devel-opment in A. flavus. In A. flavus, both laeA and veA deletion lead toloss of sclerotia production as well as reduction in conidiation (Dur-an et al., 2007; Kale et al., 2008). Moreover, the deletion of both veAand laeA also appears to disrupt quorum sensing in A. flavus as willbe discussed further in Section 4 (Amaike and Keller, 2009).

On host seeds, veA and laeA deletion strains are characterized byreduced pathogenicity. Both DlaeA and DveA strains of A. flavusproduced fewer conidia (DveA far less than DlaeA strains) and noaflatoxin on maize and peanut seeds. Furthermore, while the DlaeAstrains could invade host cells intracellularly, hyphae of DveAstrains could only grow intercellularly in epidermal cells. Boththe DlaeA and DveA strains of this fungus had significantly im-paired ability to degrade host cell lipid reserves when comparedto wild type strains (Amaike and Keller, 2009). This may be relatedto an earlier study that showed lipase gene expression was corre-lated with pathogenicity (Yu et al., 2003).

Recently, it was shown that laeA deletion in A. flavus impactsconidial hydrophobicity such that the DlaeA strains had morehydrophilic conidia (Chang et al., 2012b). This could potentially belinked to loss of aflatoxin production in DlaeA strains as the reducedhydrophobicity could impact formation of vesicles, which have beenshown to be important in aflatoxin synthesis and transport inA. parasiticus (Chanda et al., 2009). Defects of laeA and veA mutantsin secondary metabolism may be partially rescued by some proteinssuch as the bZIP protein RsmA, which in A. nidulans laeA and veA mu-tants restores sterigmatocystin biosynthesis through induction ofaflR and aflS (Shaaban et al., 2010; Yin et al., 2012, 2013).

in three Aspergillus species. Yes = has an impact on specific process in the species

Sources

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)

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)

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)

M.G. Amare, N.P. Keller / Fungal Genetics and Biology 66 (2014) 11–18 15

4. Quorum sensing in A. flavus

Filamentous fungi follow a programmed developmental se-quence from spore germination to vegetative hyphae, which candifferentiate into either sexual or asexual sporulation structures.Accumulating evidence has shown that fungi regulate develop-ment, and more recently secondary metabolism, through quorumsensing, a density-dependent phenomenon that leads to a coordi-nated population-wide response (Albuquerque and Casadevall,2012). Most studies have focused on yeast and the basidiomyceteCryptococcus neoformans with signaling molecules ranging fromsmall alcohols to various fatty acids.

A. flavus reproduces in a density-dependent manner where lowpopulation densities are characterized by increased sclerotia pro-duction and reduced conidiation (Brown et al., 2009; HorowitzBrown et al., 2008). As the population transitions to high cell den-sity, the reverse phenotype is observed with reduced sclerotia pro-duction and increased conidiation. Moreover, A. flavus alsoresponds to spent-medium extracts producing more conidia orsclerotia when exposed to high- or low-density spent-medium ex-tracts respectively. In addition to development, secondary metab-olism is also regulated in a cell density-dependent manner whereaflatoxin 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 importantin quorum sensing in A. flavus. Chief among these are oxylipins, agroup of diverse oxygenated polyunsaturated fatty acids that actas molecular signals across the fungal, plant and animal kingdoms.Collectively referred to as psi (precocious sexual inducer) factors inAspergillus species, oxylipins are known to regulate the balance be-tween asexual and sexual development in Aspergillus speciesincluding A. flavus (Calvo et al., 1999, 2001). In A. nidulans, whenoxylipin-encoding dioxygenase genes (ppo genes) were deleted,the balance between sexual and asexual development was dis-rupted (Tsitsigiannis and Keller, 2007). Similarly, simultaneous dis-ruption of A. flavus ppo genes (ppoA, ppoB, ppoC and ppoD) togetherwith another oxylipin-generating gene, lox, caused disruption inthe normal morphological development and abolished the switchfrom sclerotia to conidia even as population density transitionedfrom low to high density. Moreover, the ppoC and lox deletion mu-tant strains also consistently produced high levels of aflatoxin atany population density (Horowitz Brown et al., 2008; Brownet al., 2009). VeA and LaeA have also been found to be importantin quorum sensing as deletion of either gene results in failure toproduce sclerotia at any density (Amaike and Keller, 2009).

Oxylipins also possibly play a role in pathogenicity, as loss of sev-eral of the oxylipin genes in A. flavus has been associated with alteredpathogenicity on host seeds. In the study discussed above, when allfour ppo genes and the lox gene were disrupted simultaneously in A.flavus, the mutant strains showed markedly reduced conidiation butincreased aflatoxin production on maize and peanut seeds (Brownet al., 2009). In another study, A. flavus ppoA, ppoB and ppoC were ex-pressed during pathogenesis on hazelnut with ppoB appearing to beexpressed exclusively during pathogenesis (Gallo et al., 2010).

Oxylipins may also potentially be involved in fungus-hostcross-communication as plant-derived oxylipins (9(S)-HpODE)can functionally substitute for fungal-derived oxylipins andstimulate sporulation (Brodhagen et al., 2008; Calvo et al., 1999).Oxylipins have also been implicated in injury-response mecha-nisms that may underlie some of the developmental response infungi (Hernandez-Onate et al., 2012) as well as ability of fungalspores to germinate (Herrero-Garcia et al., 2011).

Until recently, how fungi perceive oxylipins was unknown, butthere is accumulating evidence that oxylipins are sensed by

G protein-coupled receptors (GPCRs). GPCR deletion mutants inA. flavus and other filamentous fungi exhibit disruption in develop-ment and secondary metabolism. A recent study investigating twoA. flavus GPCRs, GprC and GprD, found that their deletion led to im-paired ability to transition from low to high cell-density pheno-types as both deletion strains produced high sclerotia andaflatoxin levels even in unfavorable high population density condi-tions (Affeldt et al., 2012).

5. Conserved Aspergillus proteins involved in morphogenesis inA. flavus

In addition to the Velvet Complex proteins and oxygenases dis-cussed above, various other proteins are also known to regulatefungal development and/or secondary metabolism in A. flavus.nsd (never in sexual development) genes, initially discovered fromA. nidulans mutants that failed to produce cleistothecia (Han et al.,1998), control development and secondary metabolism in A. nidu-lans (Han et al., 2001). Two nsd genes, both encoding for GATA-typetranscription factors, act similarly in A. flavus with nsdC and nsdDdeletion mutants showing reduced conidiation (arising from aber-rant conidiophore morphology), loss of sclerotia formation and lossof aflatoxin production (Cary et al., 2012).

Another gene, fluG, which in A. nidulans is required for conidia-tion and sterigmatocystin production, was found to affect develop-ment but not secondary metabolism in A. flavus. A. nidulans fluGmutants are completely impaired in conidia production (Lee andAdams, 1994) because the mutants are unable to produce a diffus-ible factor required for sporulation that was recently characterizedto be an adduct of the meroterpenoids dehydroaustinol and dior-cinol (Rodriguez-Urra et al., 2012). A. nidulans fluG deletion mu-tants are also unable to biosynthesize sterigmatocystin (Hickset al., 1997). In contrast, in addition to highly increased sclerotiaproduction, A. flavus fluG mutants only exhibit a delay and reduc-tion in conidia production and no impact on aflatoxin production(Chang et al., 2012a). FluG potentially modulates sclerotia produc-tion through interaction with VelB as the two proteins have beenshown to interact. Moreover, fluG deletion in DvelB strains furthersignificantly decreases conidiation even in conidiation-inducinglight environment (Chang et al., 2013).

FluG interacts with a conserved G protein signaling system link-ing asexual sporulation with secondary metabolism. Briefly, FluGactivates a heterotrimeric G protein complex that ultimately signalsthrough protein kinase A (PkaA) to activate AflR (Shimizu et al.,2003). Although this system was first worked out in A. nidulans (re-viewed in Brodhagen and Keller, 2006; Park and Yu, 2012), severalof 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 anddevelopment

Oxidative stress response and secondary metabolism arethought to be highly integrated processes. Many filamentous fungi,including A. flavus, A. parasiticus, A. oryzae and A. nidulans, exhibit aclose association and interplay between these two processes wheresecondary metabolism is often induced as a response to cellularoxidative stress (for more in-depth reviews on the linkage betweenoxidative stress response and secondary metabolism and develop-ment, the reader is referred to Hong et al., 2013a; Montibus et al.,2013).

Various proteins, many belonging to the bZIP transcriptionfactor family, coordinate this interplay between oxidative stressand secondary metabolism. bZIP proteins such as AtfB, AtfA andAP-1 (and its orthologs ApYapA and NapA) play key roles in

16 M.G. Amare, N.P. Keller / Fungal Genetics and Biology 66 (2014) 11–18

co-regulation of these two processes in many filamentous fungi. InA. parasiticus, not only does atfB expression correlate with aflatoxinproduction, but AtfB also binds to promoters of aflatoxin biosyn-thesis genes as well as stress response genes (Hong et al., 2013b;Roze et al., 2011). Moreover, accumulation of aflatoxin biosynthe-sis gene transcripts as well as accumulation of atfB transcript oc-curs concurrently during aflatoxin production (Roze et al., 2011).In further evidence of the co-regulation of oxidative stress and sec-ondary metabolism, AtfB and another oxidative stress response-re-lated bZIP protein, AP-1 (an ortholog of ApYapA), heterodimerize topromote transcription of nor-1, an aflatoxin biosynthesis gene(Roze et al., 2011). Furthermore, when ApyapA was deleted inA. parasiticus, the mutants were characterized by increased suscep-tibility to extracellular oxidants. The mutants also exhibited in-creased formation of precocious reactive oxygen species andincreased aflatoxin production (Reverberi et al., 2008). In an earlierrelated study, DApyap1 strains of A. parasiticus exhibited increasedpathogenicity on maize seeds with earlier and more production ofaflatoxins (Reverberi et al., 2007). In A. nidulans, when the ApyapAhomolog, napA, was overexpressed, mutants showed more robustresistance to oxidative stress and decreased secondary metabolismsynthesis (Yin et al., 2013). To a lesser extent, another bZIP protein,AtfA, potentially also co-regulates oxidative stress and secondarymetabolism. In A. nidulans, AtfA is known to regulate oxidativestress response (Balázs et al., 2010), while an A. nidulans AtfAortholog in the plant pathogen Botrytis cinerea (BcAtf1) regulatessecondary metabolism as its deletion (Dbcatf1) leads to muchhigher accumulation of B. cinerea secondary metabolites (Temmeet al., 2012).

Aside from bZIP proteins, other proteins such as MsnA and SrrAalso co-regulate oxidative stress response and secondary metabo-lism. MsnA (ortholog of multi-stress response S. cerevisiae Msn2protein) is a zinc-finger, stress-related protein that when deleted(DmsnA) leads to increased biosynthesis of secondary metabolitesaflatoxin and kojic acid in both A. parasiticus and A. flavus. More-over, the DmsnA strains of both A. flavus and A. parasiticus showedincreased production of reactive oxygen species (Chang et al.,2011). Recently, a novel conserved motif in the promoter regionsof both aflatoxin biosynthesis and oxidative stress response geneswas discovered and the transcription factor protein SrrA (an ortho-log 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 fun-gal life; they coordinate oxidative stress response, secondarymetabolism as well as development. The bZIP proteins discussedabove, AtfA, AtfB and ApYapA and its homolog NapA, are knownto confer reactive oxygen species tolerance to conidia and/or hy-phae (Hong et al., 2013a; Montibus et al., 2013; Sakamoto et al.,2008). While not examined for oxidative stress responses, otherbZIP 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 secondarymetabolite production (Xiao et al., 2010). Another bZIP protein,the nitrogen-regulatory MeaB protein, is important in regulatingvirulence and secondary metabolism. When overexpressed,OE::meaB A. flavus strains exhibited phenotypes of decreased hostseed colonization, reduced lipase activity and loss of aflatoxin syn-thesis mirroring the phenotype of DlaeA strains (Amaike et al.,2013b). Also, as noted in Section 3, RsmA is important in sterigmat-ocystin synthesis in A. nidulans.

7. Conclusion

Over 50 years of intense research has revealed much about thegenes, molecules and factors that control the intricate process of

aflatoxin biosynthesis in A. flavus, with newer studies revealingpathways and molecules important for development, pathogenesisand secondary metabolism. The current data supports a multifac-torial complex underlying virulence which involves production ofmany secondary metabolites – not just aflatoxin, Velvet Complexmembers VeA and LaeA, a G protein-PkaA signaling pathway, sev-eral stress response systems and a quorum sensing mechanism.

It is likely that our molecular understanding of this pathogenwill accelerate over the next few years due to the availability offungal genome sequence that has allowed for coverage of geneexpression over various growth conditions including pathogenesis,life cycle progression and stress responses leading to discovery ofsecondary metabolites and other molecules important to fungalbiology (Brakhage and Schroeckh, 2011; Chiang et al., 2008,2009; Kale et al., 2008; Lim et al., 2012; Martens-Uzunova andSchaap, 2009; Schneider et al., 2007; Wang et al., 2010). Whilein-depth coverage of all of the microarray and RNAseq studies isbeyond the goal of this review; the reader is directed to specific pa-pers which have added valuable information on genes and path-ways contributing to A. flavus virulence and development (Caryet al., 2007; Georgianna et al., 2010; Gibbons et al., 2012; Linet al., 2013; Olarte et al., 2012; Reese et al., 2011; Yu et al.,2011). We hope that analysis of these studies will lead to discoveryof genes and molecules involved in pathogenicity or fungal survivalthat may contribute to efforts to control diseases caused by thisunique species, not just as a plant pathogen but also as its role asan opportunistic human pathogen.

Acknowledgment

We thank Katharyn J. Affeldt for critical commentary on thisreview.

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