SEGUNDA LECTURA El Señor corrige a los que ama

Regulation of secondary metabolite synthesis is composed of complex hierarchical cross-feeding pathways. Our knowledge of AF and ST regulatory factors has greatly expanded in the last 10 years, as considered below (Yin and Keller2011).

A. AflR

There are several Zn(II)₂Cys₆ transcription factors involved in secondary metabolite regu-lation in Aspergillus and other fungi(Yin and Keller2011). The genes for these types of tran-scription factors are typically found embedded in secondary metabolite clusters, where the encoded protein acts to activate the other genes in the cluster. The AF/ST Zn(II)2Cys6

factor, AflR, has been studied for about 20 years, and was instrumental in elucidating the mechanism of ST/AF regulation and produc-tion. AflR (formerly called Afl-2 or Apa-2) binds the motif 5⁰-TCG(N)₅CGA that is located within most promoters of ST/AF cluster genes (Chang et al. 1993; Payne et al. 1993; Brown et al. 1996; Fernandes et al. 1998, Fig. 3.1).

AflR also weakly binds a non-consensus site in its own promoter in A. parasiticus and A. flavus (Ehrlich et al. 1999). Microarray data of wild-type A. parasiticus and aDaflR mutant showed that AF cluster genes were downregulated in the DaflR strain compared to wildtype (Price et al.

2006). AflR is both transcriptionally and post-transcriptionally regulated by protein kinase A (Shimizu and Keller2001; Shimizu et al.2003).

Sharing a promoter with aflR is aflS (aflJ).

The function of AflS is unclear, but it could act as an enhancer for AflR regulation of ST/AF cluster genes (Chang2003). Disruption of aflS in A. flavus results in a loss of AF production, as

well as an inability to convert exogenous pre-cursors to AF, suggesting that aflS is required for AF biosynthesis (Meyers et al.1998). More recently, RNA-Seq data indicated that AF clus-ter genes, including aflR and aflS, were expressed much higher at 30
C than at 37
C, suggesting that temperature affects AF produc-tion via these regulators (Yu et al.2011).

B. bZIP Transcriptional Factors

Another type of transcription factor recently associated with secondary metabolite regula-tion, including AF and ST, is the basic leucine zipper domain (bZIP) protein associated with stress responses, development, and metabolite biosynthesis in many fungi (Rodrigues-Pousada et al. 2010). These transcription fac-tors contain two major motifs: a basic region, which facilitates sequence-specific DNA bind-ing, and a leucine zipper region, which allows dimerization of bZIP proteins (Fernandes et al.

1997). bZIPs can be both positive and negative regulators.

atfB encodes a bZIP transcription factor belonging to the cAMP response element (CRE) binding protein family. Microarray data in A. oryzae revealed that several stress response genes, including a catalase gene and trehalose biosynthesis genes, were downregu-lated in aDatfB mutant compared to the wild-type. TheDatfB strain germinated similarly to wildtype in stress-free conditions, but DatfB conidia were much more susceptible to heat-shock and H₂O₂stresses (Sakamoto et al.2008).

Recently, chromatin immunoprecipitation (ChIP) in A. parasiticus revealed that AtfB binds to the promoters of seven genes in the AF cluster, all of which contain CRE sites, under AF-inducing, but not AF-repressing con-ditions, suggesting AtfB to be a positive regula-tor of AF gene expression (Roze et al.2011b).

Interestingly, binding at these sites was nearly absent in a strain lacking veA, a global regulator of secondary metabolism and fungal develop-ment, and a member of the Velvet Complex (see discussion below). Electrophoretic mobility shift analysis (EMSA) confirmed that AtfB is part of a protein complex that binds to the

aflD (formerly called nor-1) promoter in the AF cluster, and that both a CRE1 and an AP-1 site are needed for binding. AP-1 is another conidial stress tolerance bZIP transcription fac-tor (Reverberi et al.2008), and this work sug-gests that AtfB may form a heterodimer with AP-1. Together, these findings revealed a link between the oxidative stress response in con-idia to production of secondary metabolites (Roze et al.2011a,b).

RsmA (remediation of secondary metabo-lism) is another bZIP transcription factor that was identified in a multicopy-suppressor screen for restoration of secondary metabolism in an A. nidulans DlaeA mutant. Overexpression of rsmA greatly increases ST synthesis (Shaaban et al. 2010). Microarray analysis of an rsmA overexpression strain showed that the entire ST cluster was upregulated in this strain (Yin et al.2012). Two putative RsmA-binding sites were identified by bioinformatic analysis, and both were found in the aflR-aflS bidirectional promoter. EMSA revealed that RsmA binds to both of these motifs, and this activates aflR to regulate ST production. One of these motifs is similar to the canonical binding site (TTAG-TAA) of a subclass of S. cerevisiae bZIP pro-teins known as YAP propro-teins, and the other is an RsmA-specific binding site, TGACACA. Not only are these sites required for RsmA binding in vitro, they are also required for aflR expres-sion and ST production in vivo (Yin et al.2012).

Another positively acting bZIP protein is meaB (methylammonium-resistant), first described in A. nidulans, where it was shown to be involved in nitrogen metabolite repression. Deletion of meaB increased colony diameter on the ammonium analog methylam-monium, and sensitivity to chlorate and nitrite with ammonium sources (Polley and Caddick 1996). MeaB activates expression of nmrA in A. nidulans, which represses nitrogen metabo-lism, by binding to a motif, TTGCACCAT, found in the nmrA promoter (Wong et al.

2007). Later work, however, showed that while MeaB may play a role in nmrA regulation, it is not its sole activator (Wagner et al. 2010). In addition, MeaB binds the same Yap-like bind-ing site as RsmA, and positively regulates ST production in A. nidulans (Amaike et al.,

unpublished). MeaB also regulates the NRPS-derived pigment metabolite, bikaverin, and the plant hormone, gibberellin in Fusarium fuji-kuroi (Wagner et al. 2010), which suggests that MeaB might bind the promoter regions of synthases or transcription factors of these clus-ters. MeaB is also an important plant pathoge-nicity factor in the vascular wilt pathogen, Fusarium oxysporum (Lo´pez-Berges et al.

2010). When meaB is overexpressed in A. flavus, it decreases conidiation on peanut seed (Amaike et al. 2013 submitted). meaB is also involved in fungal development. In A. nidulans, overexpression of meaB decreased colony diameter and conidiation, while disruption of meaB increased conidiation and decreased cleistothecia formation, suggesting that MeaB may be binding to the promoter region of asex-ual and sexasex-ual development regulators, and inducing feedback mechanisms (S. Amaike and N.P. Keller, unpublished data). These data indicate that MeaB is a regulator of secondary metabolism, nitrate utilization, plant pathoge-nicity, and fungal development through the binding-specific sites in the fungal genome.

A. parasiticus ApyapA and A. ochraceus Aoyap1 are orthologous bZIPs associated with negative regulation of AF and the mycotoxin ochratoxin respectively (Reverberi et al. 2007, 2008,2012). Both proteins are orthologs of A.

nidulans NapA, characterized for its role in protecting the fungus from oxidative stress (Asano et al. 2007). Deletion of ApyapA and Aoyap1 increases oxidative stress and AF/

ochratoxin levels in the two fungi. Here, the authors suggest that these bZIPs are required for proper redox balance in the cell, and loss of this balance stimulates AF/ochratoxin levels.

C. Velvet Complex

As mentioned, laeA (loss of aflR expression) regulates many secondary metabolites. LaeA is a member of a heterotrimeric nuclear complex called the Velvet Complex, along with two other proteins, the aforementioned VeA and

VelB(Bayrum et al.2008). The Velvet Complex is a conserved fungal-specific transcriptional regulator of several fungal processes including secondary metabolism, spore development, and stress responses to the environment (Bayrum et al. 2008; Baba et al. 2012; Wiemann et al.

2010; Wu et al. 2012). Little is known about the role of VelB in this complex, but VeA is required for production of cleistothecia, or sex-ual fruiting bodies, in A. nidulans (Kim et al.

2002), as well as sclerotia, overwintering struc-tures in both A. parasiticus (Calvo et al.2004) and A. flavus (Duran et al. 2007; Amaike and Keller2009). Moreover, VeA regulates the same set of secondary metabolites as LaeA in A.

nidulans and A. flavus (Bok and Keller 2004;

Kale et al.2008; Amaike and Keller2009). Dele-tion of either VeA or LaeA eliminated AF/ST production, whereas overexpression increases AF production (ST not assessed, Bok and Keller 2004; Kale et al.2008; Amaike and Keller2009).

VeA and LaeA orthologs have been found in various fungi, such as Fusarium verticillioides, Magnaporthe grisea, and Cochliobolus hetero-strophus(Li et al.2006; Calvo2008; Wu et al.

2012). Both LaeA and VeA also play a key role in pathogenicity in both plant and animal pathogens (Amaike and Keller2009; Bok et al.

2005; Myung et al.2012; Wiemann et al.2010;

Wu et al.2012).

VeA interacts with at least one other protein in addition to LaeA and VelB, FphA. FphA is a phytochrome that acts as a red-light receptor that represses sexual devel-opment in A. nidulans in red-light conditions (Blumen-stein et al.2005). It forms a protein complex with light response proteins LreA and LreB. LreA and LreB are orthologous to white collar proteins WC-1 and WC-2, which are key for sensing blue light in Neurospora crassa (Purschwitz et al.2008). Thus, the Velvet Com-plex appears to be the link between light sensing, fungal development, and secondary metabolism. (Calvo2008)

Although laeA-mediated regulation of sec-ondary metabolism has been shown to occur at the transcriptional level, the mechanism of LaeA has not yet been elucidated (Bok and Keller2004; Kale et al.2008; Amaike and Keller

2009). LaeA is a nuclear protein and a putative methyltransferase containing an S-adenosylmethionine (SAM) binding site required for function (Bok et al. 2006). LaeA activity is associated with epigenetic mechan-isms, where LaeA-regulated regions of the genome display heterochromatin marks when LaeA is absent but euchromatin marks when LaeA is activated (Reyes-Dominguez et al.

2010). Loss of LaeA can be partially remediated by deletion/inactivation of heterochromatin gatekeepers (Shwab et al.2007; Lee et al.2009).

D. Chromatin

The first inkling that chromatin regulation may be part of the hierarchical pathways regulating AF and ST synthesis came from a study in A.

parasiticus where placement of an AF cluster gene in another part of the genome resulted in abberant regulation of this gene (Chiou et al.

2002). Later, the identification of LaeA as a methyltransferase with similarities to histone methytransferases, and the fact that removal of AflR from the ST cluster released it from LaeA regulation (Bok et al. 2006), coupled with similar observations of AflR rescue of AF cluster silencing by ectopic placement of AflR in A. flavus (Smith et al.2007), strengthened the notion that AF and ST clusters were regulated in part through chromatin activation. Roze et al. (2007) also found that the spread of his-tone H4 acetylation paralleled the order of tran-scriptional activation of genes in the AF cluster.

Since this time, a series of studies aimed at activating chromatin through either genetically manipulating genes encoding enzymes modify-ing histone charge through acetylation and methylation, or by growing fungal cultures with epigenetic modifiers, has clearly sup-ported a role for heterochromatin/euchroma-tin control of fungal secondary metabolism (Shwab et al.2007and reviewed in Palmer and Keller 2010; Strauss and Reyes-Dominguez 2011). A role for chromatin regulation of gene expression has also been described for the trichothecene family toxin, dioxynivalenol (DON) in Fusarium graminearum (Reyes-Dominguez et al.2012).

E. Host-Microbe Interactions

Several plant metabolites have been impli-cated in AF gene regulation. The compounds receiving the most study are oxygenated fatty acids called oxylipins. Oxylipins are produced in all organisms. In fungi, they mediate devel-opment and production of secondary metabo-lites, and in plants, they are key to development and environmental adaptation. Moreover, they are critical signaling molecules in fungal/host interactions (reviewed in Christensen and Kolomiets 2011). Fungal oxylipins are pro-duced by oxygenases including Ppo and Lox enzymes, and oxygenase mutants in A. flavus and A. nidulans affect the fungus’ ability to sporulate, produce AF and ST, and colonize seed. For example, an A. nidulans DppoABC mutant was unable to produce ST in growth medium and in planta. The DppoABC mutant was also extremely impaired in its ability to colonize peanuts, showing a drastic reduction of asexual and sexual sporulation compared to the wildtype (Tsitsigiannis and Keller2006). In a reciprocal fashion, maize lipoxygenase Zmlox3 null mutant corn kernels are more resistant to several fungal pathogens (Gao et al. 2007), but are more susceptible to both A. nidulans and A. flavus, seen by an increase in conidiation and AF/ST production (Gao et al.

2009).

Plant oxylipins appear to mimic endoge-nous oxylipin signals to affect development of A. flavus, with various exogenously applied plant oxylipins altering sporulation, mostly to increase conidiation (Calvo et al. 1999). The plant lipoxygenase (13-Lox) product 13(S)-HPODE decreases ST in A. nidulans and AF in Aspergillus parasiticus but 9(S)-HPODE production by plant 9-Lox stimulate AF/ST synthesis (Burow et al. 1997). Furthermore, maize-derived Zmlox3 (a 9(S)-HPODE pro-ducer) expressed in A. nidulans causes a dramatic increase in conidiation, ST produc-tion, and cleistothecia size (Brodhagen et al.

2008). Several studies show that infection of plant seeds with Aspergillus species impacts host lipoxygenase expression (Burow et al.

2000; Wilson et al. 2001; Tsitsigiannis et al.

2005). This induction is, in part, mediated by

fungal oxylipins, as A. nidulans ppo-deletion mutants were no longer able to induce plant Lox expression, demonstrating the crosstalk between fungal and plant oxylipins (Brodhagen et al.2008). This impact of oxylipins on AF and ST synthesis has been associated with oxidative stress levels in the fungus, in part regulated by the bZIP protein Apyap discussed above.

Oxylipin production has also been implicated in quo-rum sensing in A. flavus. Quoquo-rum sensing, originally described in bacteria, is now known to exist in fungi as well. Horowitz Brown et al. (2008) demonstrated an A.

flavus conidia–sclerotia morphology shift dependent on cell density, with increasing conidial production at high cell densities. In addition to morphological altera-tions, the profile of secondary metabolites changed as a consequence of cell densities, with decreased AF syn-thesis at high cell densities (Brown et al. 2009). A critical property associated with quorum-sensing was demonstrated in A. flavus, when it was shown that high cell density extracts induced high cell density develop-ment, and vice versa. As deletions in A. flavus ppo and lox genes block the developmental shift (as does exoge-nous addition of various oxylipins), it has been pro-posed that oxylipins may serve as quorum-sensing signals in Aspergillus spp. adding yet another layer of complexity in cross-kingdom communications. (Brown et al.2009)

IV. Conclusions

The specter of disease and economic costs caused by AF contamination of food and feed crops continues to drive studies in Aspergillus research. Great advances have been made in understanding the complex genetic regulation of AF/ST in the last 10 years, enhanced by the sequence completion of several Aspergillus genomes including those of A. flavus and A.

nidulans. Insights into chromatin regulation and plant signals, which are important in acti-vating and repressing AF synthesis, provide platforms for new methodologies to control production of this potent carcinogen.

AcknowledgementsFunding has been provided for this research from the USDA Cooperative State Research, Education, and Extension Service (CSREES) project (WIS01200) and NSF IOB-0544428, sub-agreement S060039 to N.P.K.

References

Amaike S, Keller NP (2009) Distinct roles for VeA and LaeA in development and pathogenesis of Asper-gillus flavus. Eukaryot Cell 8:1051–1060

Amaike S, Keller NP (2011) Aspergillus flavus. Ann Rev Phytopathol 49:107–133

Amaike S, Affeldt KJ, Yin WB, Franke S, Choithani A, Keller NP (2013) The bZIP protein MeaB mediates virulence attributes in Aspergillus flavus (submitted)

Asano Y, Hagiwara D, Yamashino T, Mizuno T (2007) Characterization of the bZip-type transcription factor NapA with reference to oxidative stress response in Aspergillus nidulans. Biosci Biotech-nol Biochem 71:1800–1803

Baba S, Kinoshita H, Nihira T (2012) Identification and characterization of Penicillium citrinum VeA and LaeA as global regulators for ML-236B production.

Curr Genet 58:1–11

Bayrum O, Krappmann S, Ni M, Bok JW, Helmstaedt K, Valerius O, Braus-Stromeyer S, Kwon NJ, Keller NP, Yu JH, Braus GH (2008) The VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320:1504–1506

Bennett JW, Chang PK, Bhatnagar D (1997) One gene to whole pathway: the role of norsolorinic acid in aflatoxin research. Adv Appl Microbiol 45:1–15 Blumenstein A, Vienken K, Tasler R, Purschwitz J,

Veith D, Frankenberg-Dinkel N, Fischer R (2005) The Aspergillus nidulans phytochrome FphA represses sexual development in red light. Curr Biol 15:1833–1838

Bok JW, Keller NP (2004) LaeA, a regulator of second-ary metabolism in Aspergillus spp. Euksecond-aryot Cell 3:527–535

Bok JW, Balajee SA, Marr KA, Andes D, Nielsen KF, Frisvad JC, Keller NP (2005) LaeA, a regulator of morphogenetic fungal virulence factors. Eukaryot Cell 4:1574–1582

Bok JW, Hoffmeister D, Maggio-Hall LA, Murillo R, Glasner JD, Keller NP (2006) Genomic mining for Aspergillus natural products. Chem Biol 13:31–37

Brodhagen M, Tsitsigiannis DI, Hornung E, Go¨bel C, Feussner I, Keller NP (2008) Reciprocal oxylipin-mediated cross-talk in the Aspergillus–seed patho-system. Mol Microbiol 67:378–391

Brown DW, Yu JH, Kelkar HS, Fernandes M, Nesbitt TC, Keller NP, Adams TH, Leonard TJ (1996) Twenty-five co-regulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidu-lans. Proc Natl Acad Sci USA 93:1418–1422 Brown SH, Scott JB, Bhaheetharan J, Sharpee WC,

Milde L, Wilson RA, Keller NP (2009) Oxygenase coordination is required for morphological transi-tion and the host-fungus interactransi-tion of Aspergillus flavus. Mol Plant Microbe Interact 22:882–894

Burow GB, Nesbitt TC, Dunlap J, Keller NP (1997) Seed lipoxygenase products modulate Aspergillus mycotoxin biosynthesis. Mol Plant Microbe Inter-act 10:380–387

Burow GB, Gardner HW, Keller NP (2000) A peanut seed lipoxygenase responsive to Aspergillus colo-nization. Plant Mol Biol 42:689–701

Calvo AM (2008) The VeA regulatory system and its role in morphological and chemical development in fungi. Fungal Genet Biol 45:1053–1061 Calvo AM, Hinze LL, Gardner HW, Keller NP (1999)

Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp. Appl Environ Microbiol 65:3668–3673

Calvo AM, Bok JW, Brooks W, Keller NP (2004) veA is required for toxin and sclerotial production in Aspergillus parasiticus. Appl Environ Microbiol 70:4722–4739

Cardwell KF, Henry SH (2004) Risk of exposure to mitigation of effects of aflatoxin on human health:

a west African example. J Toxicol 23:217–247 Chang PK (2003) The Aspergillus parasiticus protein

AflJ interacts with the aflatoxin pathway-specific regulator AflR. Mol Genet Genomics 268:711–719 Chang PK, Cary JW, Bhatnagar D, Cleveland TE,

Bennett JW, Linz JE, Woloshuk CP (1993) Cloning of the Aspergillus parasiticus apa-2 gene associated with the regulation of aflatoxin biosynthesis. Appl Environ Microbiol 59:3273–3279

Chang PK, Horn BW, Dorner JW (2009) Clustered genes involved in cyclopiazonic acid production are next to the aflatoxin biosynthesis cluster in Aspergillus flavus. Fungal Genet Biol 46:176–182 Chiou CH, Miller M, Wilson DL, Trail F, Linz JE (2002)

Chromosomal location plays a role in regulation of aflatoxin gene expression in Aspergillus parasiti-cus. Appl Environ Microbiol 68:306–315

Christensen SA, Kolomiets MV (2011) The lipid lan-guage of plant-fungal interactions. Fungal Genet Biol 48:4–14

Cleveland TE, Yu J, Fedorova ND, Bhatnagar D, Payne GA, Nierman WC, Bennett JW (2009) Potential of Aspergillus flavus genomics for applications in biotechnology. Trends Biotechnol 3:151–157 Cole RJ, Cox RH (1981) In: Cole RJ, Cox RH (eds)

Handbook of toxic fungal metabolites. Academic, New York, pp 67–93

Crawford JM, Thomas PM, Scheerer JR, Vagstad AL, Kelleher NL, Townsend CA (2008) Deconstruction of iterative multidomain polyketide synthase func-tion. Science 320:243–246

Dereszynski DM, Center SA, Randolph JF, Brooks MB, Hadden AG, Palyad KS, McDonough SP, Messick J, Stokol T, Bischoff KL, Gluckman S, Sanders SY (2008) Clinical and clinicopathologic features of dogs that consumed foodborne hepatotoxic afla-toxins: 72 cases (2005–2006). J Am Vet Med Assoc 232:1329–1337

Duran RM, Cary JW, Calvo AM (2007) Production of cyclopiazonic acid, aflatrem, and aflatoxin by Aspergillus flavus is regulated by veA, a gene

necessary for sclerotia formation. Appl Micobiol Biotechnol 73:1158–1168

Ehrlich KC (2009) Predicted roles of the uncharacter-ized clustered genes in aflatoxin biosynthesis. Tox-ins 1:37–58

Ehrlich KC, Montalbano BG, Cary JW (1999) Binding of

Ehrlich KC, Montalbano BG, Cary JW (1999) Binding of