Signalling Pathways Connecting Mycotoxin Production and Sporulation
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MOLECULAR PLANT PATHOLOGY (2006) 7(4), 285–301 DOI: 10.1111/J.1364-3703.2006.00338.X ReviewBlackwell Publishing Ltd Signalling pathways connecting mycotoxin production and sporulation MARION BRODHAGEN AND NANCY P. KELLER* Department of Plant Pathology, University of Wisconsin—Madison, 1630 Linden Dr, Madison, WI 53706-1598, USA Claviceps purpurea (e.g. reviewed by Bennett and Klich, 2003). SUMMARY Mycotoxin contamination of food and feed presents a serious Mycotoxin contamination of food and feed presents a serious food safety issue on a global scale; therefore, agricultural food safety issue on a global scale, causing tremendous yield and commodities traded nationally and internationally are screened economic losses. These toxins, produced largely by members of and regulated for certain mycotoxins. Although it is impossible to the genera Aspergillus and Fusarium, represent a subset of the determine the actual economic losses due to mycotoxin contam- impressive array of secondary metabolites produced by fila- ination of crops, a recent estimate puts annual losses at between mentous fungi. Some secondary metabolites are associated $418 million and $1.66 billion to agribusiness in the United temporally and functionally with sporulation. In Aspergillus States (Robens and Cardwell, 2005; Vardon et al., 2003). Among and Fusarium, sporulation and mycotoxin production are both the most economically damaging mycotoxins are aflatoxins, regulated by G protein signalling pathways. G protein signalling produced by Aspergillus flavus, Aspergillus parasiticus and related pathways commonly regulate fungal development, stress response species (e.g. Goto et al., 1996); tricothecenes, including deoxyni- and expression of virulence traits. In addition, fungal development valenol (also known as vomitoxin) and T-2 toxin, produced is influenced by external factors. Among these are lipids, and in primarily by Fusarium species (Miller, 2002); fumonisins, produced particular, oxylipin signals, which may be derived from either the primarily by Fusarium spp. or Alternaria (Miller, 2002); zearalenone fungus or infected seeds. Regardless of origin, oxylipins have produced by Fusarium spp. (Miller, 2002); and ochratoxin pro- the potential to elicit profound changes in both sporulation and duced by Aspergillus and Penicillium spp. (Niessen et al., 2005). mycotoxin production in the fungus. Signal transduction via G The function of the vast majority of these metabolites for the protein signalling pathways represents one mechanism by producing organism remains enigmatic. However, intensive study which oxylipin signals might elicit these changes. Therefore, in of these toxins has yielded significant information regarding the this review we integrate discussion of oxylipin signals and of regulation of their production. Many excellent reviews exist that G protein signalling cascades as regulators of fungal development. address the biochemistry, physiology and genetics (e.g. Keller et al., 2005; Payne and Brown, 1998; Sweeney and Dobson, 1998; Yu, 2004), molecular regulation (e.g. Calvo et al., 2002; Sweeney and Dobson, 1999; Yu and Keller, 2005), and host genetics and cultural practices (e.g. Brown et al., 2004; Munkvold, 2003) INTRODUCTION affecting mycotoxin production. Here we will focus on signalling Fungi are prodigious producers of secondary metabolites, some pathways connecting sporulation with mycotoxin production, with great significance to society as antibiotics or toxins. Myco- and discuss advances in our understanding of the role of lipids in toxins can be defined as small organic molecules produced by these processes. filamentous fungi that ‘evoke a toxic response when introduced in low concentration to higher vertebrates and other animals by SIGNAL TRANSDUCTION a natural route’ (Bennett, 1987). The oldest recognized example of mycotoxin poisoning is ergotism, caused by ingestion of Pathway-specific regulation of mycotoxin production grain contaminated with ergot alkaloids from the phytopathogen One remarkable property of secondary metabolites produced *Correspondence: Tel.: +1 608 262 9795; Fax: +1 608 263 2626; by fungi (including phytotoxins, antibiotics, pharmaceuticals and E-mail: [email protected] mycotoxins) is that the genes involved in their biosynthesis and © 2006 BLACKWELL PUBLISHING LTD 285 286 M. BRODHAGEN AND N. P. KELLER regulation are frequently clustered, much as they are in prokary- as yet unknown mechanism (R.A. Butchko & N.P. Keller, unpublished otes (reviewed in Keller and Hohn, 1997; Zhang et al., 2004). This data; Meyers et al., 1998). One study (Chang, 2003) provides clustering has been demonstrated for the biosynthetic pathways evidence for a physical interaction between AflJ and AflR. of the ergot alkaloids of C. purpurea (Tudzynski et al., 1999), The significance of biosynthetic gene clusters in filamentous ochratoxin from Penicillium nordicum (Karolewiez & Geisen, fungi has long been debated. One hypothesis is that they repre- 2005), tricothecenes, zearalenone and fumonisins in Fusarium sent an extended form of selfish genes, facilitating simultaneous spp. (e.g. Hohn et al., 1993; Kim et al., 2005; Proctor et al., mobilization of a discrete biosynthetic function for horizontal 2003), and aflatoxin/sterigmatocystin production in the Aspergilli transfer (Walton, 2000). Another rationalization is that clustering (reviewed in Yu et al., 2004b; Zhang et al., 2004). The genome is associated with co-regulation of genes within the cluster, sequence of Aspergillus nidulans and three other Aspergilli, reminiscent of operons and regulons in prokaryotes (reviewed in A. flavus, A. orzyae and A. fumigatus (Galagan et al., 2005; Zhang et al., 2004). However, eukaryotes effectively orchestrate Machida et al., 2005; Nierman et al., 2005) revealed the poten- the functioning of many biosynthetic pathways for which the tial existence of approximately 40 secondary metabolite gene various genes are dispersed throughout the genome. Recently, clusters in each one of these species (Keller et al., 2005). Most a novel protein was described that lends new credence to the identified clusters encode unknown metabolites, but the afla- regulation-by-clustering hypothesis. The nuclear protein LaeA is a toxin (A. flavus/A. oryzae) and sterigmatocystin (A. nidulans) predicted protein methyltransferase that acts as a global reg- gene clusters are exceptionally well studied (Keller et al., 2005). ulator of secondary metabolite gene expression in Aspergillus These latter two will be described briefly as representing spp. (Bok and Keller, 2004; Bok et al., 2005). Genome-wide tran- mycotoxin gene clusters. script analyses via microarray comparisons between wild-type The sterigmatocystin biosynthetic gene cluster contains 25 A. nidulans and a ∆laeA strain showed transcriptional differences co-regulated transcripts, encoding at least 21 enzymatic steps, that were precisely demarcated by the predicted or known within a 60-kb region of chromosome IV of A. nidulans (Brown borders of secondary metabolite gene clusters (Bok et al., 2006). et al., 1996; reviewed in Yu et al., 2004b). Sterigmatocystin is the The aflatoxin and sterigmatocystin pathways are among the sec- penultimate precursor for aflatoxin. Reflecting this, the sterigma- ondary metabolite gene clusters under the control of the global tocystin gene cluster in A. nidulans and the ∼75-kb aflatoxin regulator, LaeA (Bok and Keller, 2004; Bok et al., 2006; N.P. Keller gene clusters of A. flavus and A. parasiticus contain homologous et al., unpublished results). Homologues of laeA also occur in genes encoding the same enzymatic functions, although gene several other genera of filamentous fungi. LaeA is notable in that, order has not been conserved (Yu et al., 2004a; reviewed in Yu unlike other global regulators of secondary metabolism (e.g. the et al., 2004b). By contrast, the gene clusters of A. flavus and Cys2His2 zinc finger global transcription factors CreA, AreA and A. parasiticus that encode aflatoxin biosynthesis are syntenic, PacC that relay environmental signals; reviewed by Yu and Keller, and the individual genes share > 95% nucleotide homology 2005), it does not appear significantly to affect growth or levels between species (Ehrlich et al., 2005; Yu et al., 1995). In strains of spore production. Secondary metabolism and sporulation are of A. oryzae, a species used by the food industry, the aflatoxin often linked in filamentous fungi, sharing some (but not all) regu- biosynthetic gene cluster can be found both in its entirety and latory elements (Adams and Yu, 1998; Calvo et al., 2002). ∆laeA fragmented (reviewed in Zhang et al., 2004). strains represent an uncoupling of these events, allowing, for the The sterigmatocystin/aflatoxin biosynthetic gene clusters first time, the study of global regulation of secondary metabolism include genes encoding two different transcriptional regulators: in a background normal for growth and development. aflR (Chang et al., 1993; Payne et al., 1993; Woloshuk et al., 1994; Yu et al., 1996a) and aflJ (Chang, 2003; Meyers et al., 1998). AflR Joint regulation of secondary metabolism and encodes a Zn(II) Cys domain protein (Woloshuk et al., 1994), 2 6 sporulation by G protein signalling which binds the palindromic sequence 5′-TCG(N5)GCA-3′ found in promoters of aflatoxin and sterigmatocystin biosynthetic genes Filamentous fungi undergo distinct life-cycle phases of growth (Ehrlich et al., 1999; Fernandes et al., 1998). In A. flavus and (accumulation of undifferentiated hyphae) and reproduction