Insights from Aspergillus Species Paul S

REVIEW ARTICLE Sexual development and cryptic sexuality in fungi: insights from Aspergillus species Paul S. Dyer & Ce´ line M. O’Gorman

School of Biology, University of Nottingham, Nottingham, UK Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 Correspondence: Paul S. Dyer, School of Abstract Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK. Tel.: Major insights into sexual development and cryptic sexuality within filamen- +44 115 9513251; fax: +44 115 9513251; tous fungi have been gained from investigations using Aspergillus species. Here, e-mail: [email protected] an overview is first given into sexual morphogenesis in the aspergilli, describing the different types of sexual structures formed and how their production is Received 12 May 2011; accepted 4 influenced by a variety of environmental and nutritional factors. It is argued September 2011. Final version published that the formation of cleistothecia and accessory tissues, such as Hu¨lle cells online 6 October 2011. and sclerotia, should be viewed as two independent but co-ordinated develop-

DOI: 10.1111/j.1574-6976.2011.00308.x mental pathways. Next, a comprehensive survey of over 75 genes associated with sexual reproduction in the aspergilli is presented, including genes relating Editor: Gerhard Braus to mating and the development of cleistothecia, sclerotia and ascospores. Most of these genes have been identified from studies involving the homothallic Keywords Aspergillus nidulans, but an increasing number of studies have now in addition cleistothecia; sclerotia; mating type; characterized ‘sex-related’ genes from the heterothallic species Aspergillus Petromyces; sexual morphogenesis; fumigatus and Aspergillus flavus. A schematic developmental genetic network is asexuality. proposed showing the inter-relatedness between these genes. Finally, the discovery of sexual reproduction in certain Aspergillus species that were formerly considered to be strictly asexual is reviewed, and the importance of these findings for cryptic sexuality in the aspergilli as a whole is discussed.

The genus Aspergillus comprises approximately 250 Introduction species (Samson & Varga, 2010) that are collectively ‘Sexuality in fungi has long been recognized as one of the termed the ‘aspergilli’. It should be noted that this num- more perplexing yet intriguing facets of the biology of ber is probably an underestimation, and conﬂicting this large and varied group of micro-organisms’ (Raper, reports exist on the actual number of accepted species 1966). Despite a further 40 years of research since John owing to the complex taxonomy of the genus (Peterson, Raper wrote this statement in his book Genetics of Sexual- 2008). Aspergillus species have traditionally been recog- ity in Higher Fungi, the process of sexual reproduction in nized by the presence of a common morphological fea- fungi is still perplexing mycologists. However, insights ture, the ‘aspergillum’, an asexual reproductive structure have been gained into many aspects of fungal sexuality consisting of a characteristic conidiophore culminating in following the application of modern molecular genetic an expanded bulbous region on which is borne phialides techniques. For example, genes involved in the determi- and metullae that generate chains of conidia (Bennett, nation of breeding systems have been identiﬁed together 2009). Phylogenetic analysis has shown that they are with a repertoire of genes involved in sexual develop- essentially a monophyletic group (Peterson, 2008). They ment, and such ‘sex-related’ genes have been used to have a ubiquitous distribution, being present in decaying

MICROBIOLOGY REVIEWS MICROBIOLOGY investigate the genetic basis of sexuality and asexuality in vegetation, soils and dust worldwide (Klich, 2002b; Ben- fungi. Many of these advances have come through or nett, 2010). The aspergilli are of particular importance have been aided by the study of Aspergillus species, which because they include in their number species that are will form the focus of the present review. highly beneﬁcial to mankind, but others that are highly

FEMS Microbiol Rev 36 (2012) 165–192 ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 166 P.S. Dyer & C.M. O’Gorman detrimental. Thus, certain species are used in food pro- Table 1. Teleomorphic genera with known Aspergillus anamorphs duction, in industry for metabolite production including Number of Homothallic : as a source of drugs, and as laboratory model organisms Genus species* Examples heterothallic for basic and applied research. By contrast, other Aspergil- Chaetosartorya 3 C. chrysella, 3:0 lus species can cause contamination of food stocks and C. cremea give rise to life-threatening infections in immunocompro- Dichotomomyces 2 D. albu, D. cejpii 2:0 mised hosts (Bennett, 2009, 2010). Emericella 43 E. nidulans, 42 : 1 The aspergilli have proved especially valuable in studies E. heterothallica of fungal sexuality because they include species with a Eurotium 64 E. herbariorum, 64 : 0 range of different reproductive modes. Some ‘mitosporic’ E. chevalieri Fennellia 3 F. ﬂavipes, F. nivea 3:0

species are only known to reproduce by asexual means Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 Neocarpenteles 1 N. acanthosporum 1:0 and have been traditionally classified in the fungal phylum Neopetromyces 1 N. muricatus 1:0 the Deuteromycota, which encompasses fungi lacking a Neosartorya 40 (46)† N. fischeri, 33 : 7 known sexual state (Taylor et al., 1999). By contrast, ‘mei- N. fumigata osporic’ species can also reproduce by sexual means and Penicilliopsis 2 P. dybowskii, 2:0 so have been traditionally classified in the fungal phylum P. flavidus the Ascomycota, whose characteristic feature is sexual Petromyces 5 P. alliaceus, 1:4 P. flavus reproduction involving the production of ascospores Sclerocleista 2 S. ornata, 2:0 within asci. In the case of meiosporic Aspergillus species, S. thaxteri they exhibit either heterothallic (obligate outbreeding) or Warcupiella 1 W. spinulosa 1:0 homothallic (self-fertile) sexual breeding systems. The *All legitimate species registered at MycoBank (www.mycobank.org) division of Aspergillus species into either the Deuteromy- on 5 May 2011. Note that the number of Aspergillus species regis- cotina or the Ascomycotina is arguably misleading because tered at MycoBank is higher than the figure reported by Samson & phylogenetic analysis shows that they form a united group Varga (2010). (Peterson, 2008), and the term ‘Deuteromycotina’ has †The number in parentheses includes six unconfirmed species pro- now largely been replaced by the mitosporic or meiosporic posed by Peterson (2008). terminology (Ku¨ck & Po¨ggeler, 2009). The majority of accepted Aspergillus species (approximately two-thirds of Overview of sexual development in the taxa) are only known to reproduce by asexual means, aspergilli whilst those that do exhibit sexual cycles are overwhelm- ingly homothallic in nature with few heterothallic species Morphology of teleomorph states described (Table 1) (Dyer, 2007; Kwon-Chung & Sugui, 2009). Elucidating the genetic basis of such differences in The Pezizomycotina encompasses those Ascomycota that reproductive mode is proving a fascinating challenge. grow by production of filamentous hyphae. In this sub- There have been a number of dedicated reviews over phylum, sexual spores (ascospores) are housed in one of the past decade dealing with different aspects of sexual four main types of fruiting body (ascomata/ascocarps): reproduction in Aspergillus species, some reviewing the cleistothecia, perithecia, apothecia or pseudothecia. They aspergilli in general (e.g. Dyer, 2007; Geiser, 2008) whilst differ in their size, shape, style and organization of the others have focussed specifically on Aspergillus nidulans asci, and presence and type of interascal sterile hyphae (e.g. Braus et al., 2002; Han et al., 2008a; Han, 2009; Han (Po¨ggeler et al., 2006). Members of the genus Aspergillus & Han, 2010). The present review will first give an over- produce their ascospores in cleistothecia, which are the view of sexual reproduction in the aspergilli as a whole only ascomatal type that fully encloses the asci and and then provide a summary of the current state of ascospores (Gre. kleistos, closed + Gre. the¯kion, small knowledge of genes involved with sexual development in case). Cleistothecia may contain up to 100 000 asci, each Aspergillus species. This includes updating information enclosing (with very rare exceptions) eight ascospores; in from previous reviews and covering new material relating the case of A. nidulans, an average cleistothecium may to recently characterized genes, and the involvement of contain around 80 000 viable ascospores (Pontecorvo, sex-related genes in sclerotial development. Finally it will 1953; Braus et al., 2002). Rather than being forcefully dis- describe the recent discoveries of sexual cycles in Aspergil- charged, ascospores are released following the natural lus fumigatus, Aspergillus flavus, Aspergillus parasiticus and breakdown of the ascus wall and the outer wall (perid- Aspergillus nomius and discuss the significance of these ium) of the cleistothecium in their natural environment findings in terms of cryptic sexuality in the aspergilli such as soil or decaying vegetation. One exception is given their economic and medical importance. A. athecius, which fails to form true cleistothecia; instead,

asci develop directly from ascogonial coils (coiled hyphae (a) (b) A containing the maternal nuclei that are fertilized at the AH AH onset of sexual reproduction) and are borne naked on FC undifferentiated mycelium (Raper & Fennell, 1965). Following conventional fungal nomenclature, when sexual reproduction occurs the sexual phase is termed the A ‘teleomorph’ state, whereas when asexual reproduction FC HC occurs the asexual phase is termed the ‘anamorph’ state. Thus, sexually reproducing species of Aspergillus will have (c) (d) AH SP two Latin binomials names, one for the anamorphic state A and one for the teleomorphic state under the rules of Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 ‘dual nomenclature’, for example A. fumigatus is the anamorphic state of the teleomorph Neosartorya fumigata AP (O’Gorman et al., 2009). Aspergillus species, as defined by their anamorphic state, are phylogenetically linked to 12 AM different teleomorphic genera (Table 1) (Peterson, 2008; FH A Samson & Varga, 2010). This diversity might appear sur- SM prising, but is attributed to the fact that the aspergilli Fig. 1. Diagrammatic representation (not to scale) of the exhibit a wide range of differing sexual fruit body mor- cleistothecial forms of the teleomorphic genera (a) Eurotium, (b) phologies, despite sharing similar asexual morphology, Emericella, (c) Neosartorya, (d) Petromyces. AH, ascogenous hyphae; and hence a variety of teleomorphic genera have been FC, flattened cells; A, asci, each containing eight ascospores; HC, erected to distinguish the different sexual states. Strictly Hu¨lle cells; FH, flattened hyphae; AM, ascocarp matrix; SM, stromal speaking, a teleomorph name (where available) should be matrix; AP, ascocarp peridium, composed of irregularly flattened cells; SP, stromal peridium, composed of pigmented, thick-walled pseudo- used in preference to the anamorph name according to parenchymatous cells. the rules of dual nomenclature (Bennett, 2010). However, it should be noted that the future application of dual nomenclature is currently under debate [see discussion in hardened, thick-walled, generally darkly pigmented and Bennett (2010), supplementary materials of O’Gorman spherical structure formed for survival under adverse con- et al. (2009), and Hawksworth et al. (2011)]. In this ditions. Cleistothecia develop within the sclerotium, review, where an anamorph name is particularly well embedded in a stroma (a mass or matrix of vegetative established [e.g. A. fumigatus (teleomorph N. fumigata) hyphae) consisting of pseudoparenchymatous hyphae and A. nidulans (teleomorph Emericella nidulans)], this called the stromal matrix. In this latter case, the peridia epithet will be referred to by convention. of the cleistothecia are composed of irregularly flattened Eight of the teleomorphic genera associated with the cells (Fig. 1d). Within these teleomorphic genera, the nat- aspergilli contain less than five species (Table 1). The four ure of ascospore ornamentation can often be used as a most common and widely distributed teleomorphic gen- species-specific taxonomic character (Samson & Varga, era are Eurotium, Emericella, Neosartorya and Petromyces, 2010), with some exquisite forms present. which are distinguished by the morphology of their cleis- In general, distinct gametangia (e.g. ascogonia and an- tothecia as illustrated in Fig. 1. One of the main differ- theridia) have not been observed in any of the aspergilli ences between them is the composition and often colour except for the genus Fennellia (Geiser, 2008). However, of the cleistothecial wall (the ‘peridium’) (Benjamin, this might be an artefact because of the overwhelming 1955; Geiser, 2008). In Eurotium, a single layer of fre- number of homothallic aspergilli, which might be expected quently yellow, large flattened cells forms the cleistothe- to exhibit reduced mating apparatus concomitant with cial wall (Fig. 1a). In Emericella, the peridium is other genomic adaptations (Paoletti et al., 2007). Evidence composed of two layers of often dark-purple flattened from both classical cytological studies and more recent cells, which in A. nidulans at least are glued together with genetic and molecular analyses indicates that nuclei from an unknown electron-dense substance (‘cleistin’) that fills one mating partner (or nucleus in a homothallic species) the intercellular spaces (Fig. 1b) (Sohn & Yoon, 2002). normally contribute entirely to the formation of maternal By contrast, the cleistothecial walls of Neosartorya consist tissues forming the accessory tissues and cleistothecial wall, of a network of layers of interlocking flattened hyphae whilst the other paternal partner merely contributes a that are normally white to light yellow in colour (Fig. 1c). compatible fertilizing nucleus that undergoes division and In the genus Petromyces, cleistothecia are formed inside passes into the ascogenous hyphae (the specialized multi- another larger structure known as a ‘sclerotium’. This is a nucleate hyphae contained within the cleistothecia where

FEMS Microbiol Rev 36 (2012) 165–192 ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 168 P.S. Dyer & C.M. O’Gorman compatible nuclei fuse to give rise to crozier tips and asci) 2010), there has been speculation that the ancestral type and ultimately the diploid zygote (Apirion, 1963; Zonne- of ascomata might have been characterized by a well- veld, 1988a; Bruggeman et al., 2003a; Todd et al., 2006). developed pseudoparenchymatous wall enclosing the asci, However, ‘mosaics’ where both partner nuclei contribute borne within a loose hyphal stroma. Divergent evolution to the cleistothecium wall have also been observed in within the aspergilli then led to a gradual reduction and A. nidulans, although only infrequently such as the special- simpliﬁcation of the peridium in some cases, together ized instance of sex developing from a preformed hetero- with either specialization or loss of stroma tissues during karyotic mycelium (Bruggeman et al., 2003a). evolution (Malloch & Cain, 1972). Indeed, in later parts Emericella is the only known teleomorphic genus that of the present review, it will be observed that some of the produces Hu¨lle cells (Ger. die Hu¨lle, casing/envelope) initial developmental pathways relating to sex, such as – l

(Raper & Fennell, 1965). These are large (c. 10 15 m Hu¨lle cell and sclerotial production, can proceed inde- Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 diameter), thick-walled, globose cells that surround the pendently of the formation of ascogenous hyphae and cleistothecia during development, with several hundred meiosis. Thus, two successive sexual developmental pro- Hu¨lle cells surrounding the cleistothecia of A. nidulans grammes are apparent in the aspergilli; the first relating for example (Sarikaya Bayram et al., 2010). They are mul- to the initial formation of supporting accessory tissues tinucleate, but interestingly at maturity a macronucleus needed for sex in some species, for example Hu¨lle cells with a volume 209 that of normal nuclei is formed in and sclerotia, and the second relating to subsequent A. nidulans, apparently from the grouping of smaller cleistothecia and ascospore production. Normally, these nuclei (Carvalho et al., 2002). The precise morphology of developmental programmes would be expected to be Hu¨lle cells varies according to species grouping and can synchronized and follow on from one another. However, be used as a valuable diagnostic character, and Hu¨lle cells sometimes they may become unlinked. This is illustrated can germinate to initiate new hyphal growth (Raper & by species that form accessory tissues independent of the Fennell, 1965). Hu¨lle cells have a specialized physiology, later stages of sex, for example Hu¨lle cell formation in exhibiting, for example, laccase and chitin synthase activ- A. multicolour, A. subsessilis, A. sydowii and A. puniceus ity (Bayram & Braus, 2011), and it has long been believed (see above); sclerotial formation in A. caelatus and that they ‘nurse’ the cleistothecia during development, A. sclerotioniger (see above); and various mutants of such as through the production of a-1,3 glucanase that A. nidulans (see later) that can produce copious amounts mobilizes carbon resources required for fruiting body of Hu¨lle cells despite the absence of meiosis and asco- development (Wei et al., 2001). Indeed, it was recently spore development. It is significant to note that members shown that a reduction in the number of Hu¨lle cells sur- of the genus Petromyces may produce from single to mul- rounding ascomata of A. nidulans to only 2–5 per cleisto- tiple cleistothecia, each with a distinct peridium, within a thecium in a DlaeA mutant (see later) resulted in single sclerotium – this fact emphasizing the indepen- significantly smaller cleistothecia, which were only 40 lm dence of the two developmental pathways. (e.g. see Fig. 3 in diameter unlike wild-type cleistothecia (c. 200 lm of Horn et al., 2011). It will be interesting to compare diameter) (Sarikaya Bayram et al., 2010). Hu¨lle cells are the developmental pathways of stromatic accessory tissues also present in some supposedly asexual species in the in the aspergilli with those seen in more traditional asco- sections Nidulantes (e.g. A. multicolour, A. subsessilis, loculate fungi (i.e. Ascomycota where the asci develop in A. sydowii) and Usti (e.g. A. puniceus) (Raper & Fennell, cavities in a preformed stroma) (Debuchy et al., 2010). 1965; Klich, 2002a). Intriguingly, their presence in these species suggests that they are vestigial remnants of sexual Environmental factors influencing sexual cycles that were presumably lost over the course of evolu- reproduction tion. Similarly, a number of supposedly asexual species in sections Flavi (e.g. Aspergillus caelatus) and Nigri (e.g. Within the aspergilli, and indeed the Pezizomycotina as a A. sclerotioniger) produce sclerotia composed of hardened whole, sexual and asexual spore production can be viewed masses of hyphae (McAlpin, 2004; Samson et al., 2004). as two competing forms of reproduction with one devel- It was previously thought that sclerotia provide an evolu- opmental process inhibiting the other and vice versa. The tionary advantage by functioning as a survival mechanism type of reproduction that occurs is largely governed by during adverse environmental conditions. However, it has environmental factors, with conidia or ascospore produc- been suggested that, under the right conditions, such scle- tion favoured as the result of a sum of environmental rotia would also be capable of acting as repositories for influences (Dyer et al., 1992). In the case of the aspergilli, cleistothecia (Rai et al., 1967; Geiser et al., 1998). there is no single set of conditions that favours sexual Given that the genus Aspergillus forms an essentially reproduction; rather this is both species and subgenus monophyletic clade (Peterson, 2008; Samson & Varga, specific and relates to the ecology of the species involved.

As most of our knowledge comes from studies of the 2003). In general, a balanced carbon/nitrogen ratio is model organism A. nidulans (as will be described below), most favourable for sex, and when suitable nitrogen it should therefore be cautioned that these findings may sources are available, sexual reproduction is preferred not be applicable to all aspergilli. over asexual sporulation (Zonneveld, 1977; Han et al., A first key factor influencing sexual development is 2003). A slight increase in glucose and decrease in nitro- the absence or presence (including wavelength) of light. gen concentration was claimed to favour cleistothecial Given that many Aspergillus species are natural soil dwell- development in A. nidulans (Swart et al., 2001). By con- ers (Klich, 2002b), the need to determine whether they trast, development of cleistothecia in xerophilic Eurotium are above or below ground can be seen as essential species occurs best at media containing up to 30% because this can favour the production of airborne coni- glucose (Blaser, 1975). Another aspect of well-nourished

dia or more environmentally resistant ascospores, respec- conditions is the presence of a suitable amino acid source(s), Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 tively. In A. nidulans, the sexual cycle can take place in with development of cleistothecia found to be blocked both the light and dark, but darkness is preferential, as specifically at the microcleistothecial stage when A. nidu- light delays initiation of the pathway by 15 h (Yager, lans is cultivated under amino acid starvation (Hoffmann 1992). This ‘light-sensitive period’ lasts from 20 to 50 h et al., 2000). The importance of phosphorus has been after inoculation. Furthermore, Zonneveld (1977) showed much less studied, but low phosphorus concentrations that incubation in darkness for 24 h directly after inocu- suppress sexual development, presumably due to a lation significantly increases cleistothecial numbers. Han requirement for phosphorus in the generation of ATP et al. (2003) found that cleistothecia were not formed in (Bussink & Osmani, 1998). Manganese is the only trace intense light and suggested that this is a stress response, element to date that has been shown to be essential for as vegetative growth is also blocked at high light intensi- cleistothecium formation in A. nidulans (Zonneveld, ties. In particular, the sexual cycle is differentially regu- 1975, 1977). A lack of manganese impairs the activity of lated by exposure to red light, blue light and far-red light, the enzyme phosphoglucomutase, which is required for with red and blue light inhibiting sexual development the synthesis of a-1,3-glucan (Zonneveld, 1972). Indeed, and far-red light enhancing development (Blumenstein A. nidulans is acleistothecial without a-1,3-glucan (Pola- et al., 2005; Bayram et al., 2008b). Meanwhile, light has check & Rosenberger, 1977). Further factors influencing also been shown to influence sclerotial formation (a sexual reproduction include the pH of the growth med- required precursor for cleistothecial development) in the ium, maximum numbers of cleistothecia in A. nidulans genus Petromyces. In general, increased numbers of sclero- being produced around neutral pH values (Rai et al., tia are produced in darkness by species including A. fla- 1967; Thakur, 1973), and the salt concentration – rela- vus and A. parasiticus (Rai et al., 1967; Bennett et al., tively high levels of salt inhibiting the sexual cycle 1978; Calvo et al., 2004; Duran et al., 2007), although although vegetative growth remains possible (Lee et al., some strains of A. flavus and black aspergilli have been 2001; Kim et al., 2009; Wang et al., 2010). Meanwhile, reported to produce equal or greater numbers of sclerotia growth medium composition also has a major influence in the light (Rai et al., 1967; Calvo et al., 1999). Light of on the induction and maturation of sclerotia in the asper- blue and green wavelengths was particularly inhibitory to gilli. For example, numbers of sclerotia produced and sclerotial development in A. parasiticus, whereas red light percentage of stroma bearing ascocarps is greatly influ- had no effect (Bennett et al., 1978). enced by the type of nitrogen source in Petromyces alliac- A second major factor determining whether sex can eus, with certain amino acids enhancing the formation of occur is the composition of the growth medium such as sclerotia but not necessarily leading to a concomitant nutrient content and pH. In the case of A. nidulans, sex- increase in numbers of ascocarps (McAlpin & Wicklow, ual development requires ‘well-nourished’ conditions for 2005). The source and concentration of carbon, nitrogen abundant cleistothecial production (Han et al., 2003), in and sulphur also impact on sclerotial production in cer- contrast to nutrient limitation that triggers sex in many tain members of the black aspergilli (Agnihotri, 1968, other pezizomycete fungi (Dyer et al., 1992). Once above 1969). Similarly, sclerotial formation by A. caelatus and a threshold carbon level, cleistothecial numbers in Aspergillus japonicus is affected greatly by the type and A. nidulans are directly related to the amount and type of concentration of carbon and nitrogen sources (Heath & carbon that is available (Zonneveld, 1974; Han et al., Eggins, 1965; McAlpin, 2004). Sclerotial development in 1994, 2003). High carbon concentrations (e.g. 6% glu- Aspergillus ochraceus is greatly affected by both the nutri- cose) inhibit sex, but this is because nitrogen then ent source and pH of the medium (Paster & Chet, 1980), becomes the limiting factor, whilst some carbon sources and sclerotial formation is pH dependent in a number of (e.g. lactose) can promote sex even in the light whereas other aspergilli (Rai et al., 1967; Thakur, 1973). Varying other sources (e.g. acetate) are inhibitory (Han et al., medium depth and water availability also has a dramatic

FEMS Microbiol Rev 36 (2012) 165–192 ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 170 P.S. Dyer & C.M. O’Gorman impact on sclerotial production in A. flavus (Thakur, is also temperature dependent, fruit bodies being observed 1973; Okuda et al., 2000; Nesci et al., 2007). Further- only at one (30 °C) of the various temperatures assayed more, attempts to induce the sexual cycle of A. fumigatus (O’Gorman et al., 2009). Similarly, highest numbers of in vitro were only successful on one particular medium cleistothecia are produced by Eurotium species between 27 (oatmeal agar) of various tried, illustrating the ‘fastidious’ and 33 °C (Blaser, 1975), and sclerotial production in nature of certain aspergilli in their sexual requirements A. flavus is temperature dependent (Rai et al., 1967). (Kwon-Chung & Sugui, 2009; O’Gorman et al., 2009). In addition, there are other factors that can influence Finally, it is noted that addition of linoleic acid can sig- the success of crosses within a laboratory setting. In the nificantly promote the production of both cleistothecia case of heterothallic aspergilli, it is becoming apparent and sclerotia in A. nidulans and A. flavus, respectively that much greater success can be achieved by inoculating

(Calvo et al., 1999; Brown et al., 2008), possibly linked to mating partners in a barrage formation rather than using Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 a role as both an energy reserve and precursor for oxyli- a single point or spread mixed spore inoculum (O’Gor- pin biosynthesis (see below) (Dyer et al., 1993; Brodhun man et al., 2009; S. Swilaiman & P.S. Dyer, unpublished & Feussner, 2011). results). And it has been found that the density of spore A third factor influencing the extent of sexual repro- inoculum used to establish Petri dish cultures has a major duction is the presence of various atmospheric gases. impact on formation of sclerotia by A. flavus and A. par- Development of cleistothecia in A. nidulans will normally asiticus, the greatest mass of sclerotia being produced only occur when grown on a medium with an air inter- using low spore concentrations in the range 101–103 face, for example an agar surface. If grown in submerged spores per plate, possibly relating to quorum-sensing oxy- liquid culture, the mycelium normally remains entirely lipin signalling (Brown et al., 2008, 2009). undifferentiated, that is, there is a fundamental influence of dimensionality (Champe et al., 1994). On solid media, Benefits of sexual reproduction cleistothecial formation in A. nidulans is significantly enhanced by elevated levels of carbon dioxide (Zonneveld, There are many potential reasons why a number of asper- 1977, 1988b). This is commonly achieved in the labora- gilli, especially facultatively sexual species such as A. nidu- tory by sealing the plates to limit air exchange. As CO2 is lans, have retained the ability to undergo sexual needed for both the synthesis and breakdown of a-1,3- reproduction despite the increased metabolic costs com- glucan, a lack of CO2 reduces cleistothecial production pared to purely asexual reproduction (Rice, 2002). Sexual (Zonneveld, 1988b). Plate sealing in addition functions to reproduction involving outcrossing (which is possible lower the oxygen concentration, causing irreversible entry even in homothallic species) can generate large amounts into the sexual cycle. This appears to arise because of the of genetic variation and produce novel genotypes much partial blockage of the electron transport system from the faster than by asexual means, as illustrated by the many lack of oxygen (Han et al., 2003). By contrast, plate seal- novel genotypes that were observed to arise from crossing ing impedes cleistothecium formation in Eurotium species strains of A. fumigatus (O’Gorman et al., 2009). This can (A. Ashour & P.S. Dyer, unpublished results), possibly increase the mean fitness of the next generation, quicken- reflecting the different ecological niches of each genus – ing adaptation to changes in the environment and xerophilic Eurotium species often being found on exposed improving their chances for long-term survival. Such an surfaces c.f. soil-dwelling Emericella species. Plate sealing increase in fitness associated with sex has been shown can also prevent the formation of sclerotia in some experimentally for A. nidulans (Schoustra et al., 2010). In strains of A. flavus and black aspergilli (Okuda et al., parallel, sex increases gene flow, bringing together and 2000; H. Darbyshir & P.S. Dyer, unpublished results). subsequently proliferating advantageous mutations as well Relative humidity (RH) also greatly influences both scle- as removing deleterious mutations that would otherwise rotia and cleistothecia production in the aspergilli, maxi- persist and accumulate in asexual populations (i.e. the mum numbers being produced at 100% RH (Rai et al., concept of ‘Muller’s ratchet’) (Dyer & Paoletti, 2005). 1967). Most recently, nitric oxide has been shown to pro- Indeed, sexual reproduction has been shown to slow mote cleistothecial production in A. nidulans (Baidya down the accumulation of deleterious mutations in et al., 2011; Marcos et al., 2011). A. nidulans, possibly through a ‘selection arena’ operating A final environmental factor influencing sexual devel- at the dikaryotic stage to favour the proliferation of opment is temperature. Traditionally, A. nidulans is incu- nuclei with higher fitness (Bruggeman et al., 2003b, bated at 37 °C to induce the sexual cycle (Pontecorvo, 2004). In addition, the ascospores of many Aspergillus 1953), whereas an almost twofold increase in the number species have a far superior ability compared to conidia to of cleistothecia can be achieved by incubation at 32 °C survive harsh environmental stresses such as desiccation (Dyer, 2007). Production of cleistothecia by A. fumigatus and high temperatures, in part because of their thickened

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 36 (2012) 165–192 Published by Blackwell Publishing Ltd. All rights reserved Sexual development and cryptic sex in the aspergilli 171 cell walls (Baggerman & Samson, 1988; Dijksterhuis, occur, whereas others appear to moderate levels of fertil- 2007). ity (‘fertility’ here used to refer to numbers of cleisto- However, it is important to note that there are also thecia produced containing mature ascospores; high many benefits to asexual reproduction in the aspergilli. fertility meaning high numbers produced and vice versa These include the ability to produce prolific numbers of for low fertility). Many of these genes have been conidia for dispersal of the species in a shorter time than described in depth in the review of Dyer (2007) to is required for ascospore production (Champe et al., which readers are referred for extra detail. An updated 1994), the lower metabolic costs associated with asexual version now follows, including details of genes identified sporulation, and the ability to produce asexual propagules since that review was written and incorporating new on a wider range of substrates and under a broader set of data from Aspergillus species not previously included.

environmental conditions than the often more fastidious These ‘sex-related’ genes are involved at various stages Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 requirements for sexual reproduction. In addition, asexual of the sexual developmental pathway, and a schematic reproduction may allow better selection and adaptation genetic network showing relationships between the vari- for local niches and limit the spread of parasitic transpos- ous genes is proposed in Fig. 2. A key principle of this able elements (Wright & Finnegan, 2001). is that ultimately sex involves bringing together two nuclei to form a diploid zygote, which then undergoes meiosis and all the ‘sexual circuitry’ works to enable this Genes involved with sexual to occur. Thus, some sets of genes are involved with development in the aspergilli environmental sensing of suitable conditions for sex; The ease with which the homothallic sexual cycle of these genes then activate the early stages of sexual devel- A. nidulans can be induced under laboratory conditions, opment with the formation of maternal hyphae with together with availability of genome sequence data and a receptive nuclei, the ascogonia. Most likely, these envi- variety of tools allowing genetic manipulation (e.g. Archer ronmental sensing genes then also activate/upregulate & Dyer, 2004; Galagan et al., 2005; Todd et al., 2007), genes controlling mating processes designed to attract has made this species an ideal subject for studies aiming and bring about the union of complementary nuclei to identify genes involved in sexual development. Indeed, with those in the ascogonia. Once fertilization and kary- almost all research efforts in this area relating to the ogamy have occurred, later signalling processes occur to aspergilli have been focussed on A. nidulans. It is there- bring about the synchronous development of the fruit fore cautioned that results from A. nidulans might not body and meiotic tissues. The precise details of the rela- necessarily be generally applicable to all aspergilli. Also, tive stages of gene action and importance of certain most studies using A. nidulans have been made with veA1 genes may vary according to whether species exhibit strains (described below), and therefore results may not homothallic or heterothallic breeding systems, but the fully reflect wild-type functionality. However, there are overall schematic model in Fig. 2 should provide a use- now also an increasing number of studies on other asper- ful guide for the aspergilli in general, as well as being gilli to substantiate results with A. nidulans, notably valid for aspects of sexual reproduction in other peziz- A. fumigatus which exhibits a heterothallic breeding sys- omycete fungi. In the following section, these sets of tem (O’Gorman et al., 2009). The latter species is there- genes are categorized under various subheadings accord- fore a more suitable organism for studying the genetic ing to their particular site of action in the sexual cycle basis of the mating process in obligate outcrossing Asper- or nature of encoded protein. gillus species, especially given that it is now possible to It is noted that other genes required for general metab- cross isolates in only 4–8 weeks (J. Sugui, C.M. O’Gor- olism may also be necessary for sexual development [e.g. man, P.S. Dyer & K.J. Kwon-Chung, unpublished results). genes for arginine, histidine, tryptophan, riboflavin and In addition, a limited number of studies have investigated phosphatidylcholine biosynthesis, and sumoylation the molecular genetic basis of sclerotial development in (Champe et al., 1994; Eckert et al., 1999; Todd et al., the aspergilli, this process now known to be a vital early 2007; Tao et al., 2010; Sarikaya Bayram et al., 2010)], but stage of sexual reproduction in Aspergillus species with these are not referred to here as these roles are arguably teleomorphs in the genus Petromyces. not specific to sex. Also, many of the genes described To date, at least 78 genes have been identified which below have been detected by BLAST analysis in the ge- are associated with sexual reproduction in A. nidulans, nomes of presumed asexual aspergilli such as Aspergillus A. fumigatus, A. flavus, A. parasiticus and most likely the oryzae, Aspergillus niger and Aspergillus terreus (Galagan aspergilli in general. Table 2 lists all genes currently et al., 2005; Dyer, 2007; Pel et al., 2007), but their func- known to have a proven role in the sexual cycle. Some tional role in these species is as yet undetermined so will of these genes are essential for sexual development to not be discussed further.

Table 2. Summary of known genes involved with sexual reproduction in the aspergilli

Gene Protein function (domain) Sex effect* Locus ID† Reference

Perception of environmental signals fphA Red light–sensing phytochrome (P2, GAF, PHY, HKD, RRD) Repressor AN9008 Blumenstein et al. (2005) lreA Blue light–sensing white-collar (LOV, PAS, zinc-finger) Activator AN3435 Purschwitz et al. (2008) lreB Blue light–sensing white-collar (PAS, zinc-finger) Activator AN3607 Purschwitz et al. (2008) cryA Blue light- and UVA-sensing cryptochrome (PHR) Repressor AN0387 Bayram et al. (2008a) veA Light/dark response (velvet complex) Activator AN1052 Kim et al. (2002) velB Light/dark response (velvet complex) Activator AN0363 Bayram et al. (2008b) velC Light/dark response Activator AN2059 Park & Yu (pers. commun.) ‡ laeA Light/dark response (methyl transferase) Bimodal AN0807 Sarikaya Bayram et al. (2010) imeB Light response, low glucose sensing (?) (TXY MAP kinase) Bimodal AN6243 Bayram et al. (2009) Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 silA Light response (zinc-finger) Repressor AN1893 Han et al. (2008c) silG Light response (zinc-finger) Repressor AN0709 Han et al. (2005) cpcA Amino acid sensing (bZIP) Repressor AN3675 Hoffmann et al. (2000) cpcB Amino acid sensing (WD repeat) Activator AN4163 Hoffmann et al. (2000) lsdA High salt sensing Repressor AN2330 Lee et al. (2001) phoA Low phosphorous sensing (cyclin-dependent kinase) Repressor AN8261 Bussink & Osmani (1998) An-pho80 Low phosphorous sensing (cyclin) Activator AN5156 Wu et al. (2004) esdC Early sexual development (glycogen binding domain) Activator AN9121 Han et al. (2008b) fhbA (fhbB) NO response (flavohaemoglobin) Repressor AN7169 (N/A§) Baidya et al. (2011)

Mating processes and signal transduction MAT1 Mating type (alpha) Activator AN2755 Paoletti et al. (2007) MAT2 Mating type (HMG) Activator AN4734 Paoletti et al. (2007) ppgA Pheromone precursor (a-type) Activator¶ AN5791 Paoletti et al. (2007) preA/gprB a-Type pheromone receptor (GPCR seven transmembrane) Activator AN7743 Dyer et al. (2003), Seo et al. (2004) preB/gprA a-type pheromone receptor (GPCR seven transmembrane) Activator AN2520 Dyer et al. (2003), Seo et al. (2004) fadA G protein a subunit Activator AN0651 Rose´ n et al. (1999) sfaD G protein b subunit Activator AN0081 Rose´ n et al. (1999) gpgA G protein c subunit Activator AN2742 Seo et al. (2005) ﬂbA Regulator of G protein signalling Activator AN5893 Han et al. (2001) phnA Phosducin-like chaperone Activator AN0082 Seo & Yu (2006) STE20 MAP kinase, kinase, kinase, kinase N/A AN5674 Dyer et al. (2003) steC/steB MAP kinase, kinase, kinase Activator AN2269 Wei et al. (2003) STE7 MAP kinase, kinase Activator¶ AN3422 Paoletti et al. (2007) mpkB MAP kinase Activator AN3719 Paoletti et al. (2007) steA Transcription factor (homoeodomain and zinc-ﬁnger) Activator AN2290 Vallim et al. (2000) ste50 Kinase cascade regulator Activator¶ AN7252 Paoletti et al. (2007) rasA Small G protein (GTPase) Repressor AN0182 Hoffmann et al. (2000) gprD GPCR (seven transmembrane) Repressor AN3387 Han et al. (2004) gprK GPCR (seven transmembrane) Activator AN7795 Yu (pers. commun.) gibB G protein (b subunit-like) Activator N/A Kong & Yu (pers. commun.) ricA GDP/GTP nucleotide exchange factor Activator N/A Kwon & Yu (pers. commun.) sakA/hogA Osmotic and oxidative stress response (MAP kinase) Repressor AN1017 Kawasaki et al. (2002) atfA Stress response (bZIP domain) Repressor AN2911 Lara-Rojas et al. (2011)

Transcription factors and other regulatory proteins stuA Transcription factor (bHLH, APSES) Activator AN5836 Wu & Miller (1997) medA Transcription factor Activator AN6230 Busby et al. (1996) devR Transcription factor (bHLH) Activator AN7553 Tu¨ncher et al. (2004) dopA Transcription factor (leucine-zipper) Activator AN2094 Pascon & Miller (2000) nsdC Transcription factor (zinc-finger) Activator AN4263 Kim et al. (2009) nsdD Transcription factor (zinc-finger) Activator AN3152 Han et al. (2001) nosA Transcription factor (zinc-finger) Activator AN1848 Vienken & Fischer (2006) rosA Transcription factor (zinc-finger) Repressor AN5170 Vienken et al. (2005) flbC Transcription factor (zinc-finger) Repressor AN2421 Kwon et al. (2010a)

Table 2. Continued

Gene Protein function (domain) Sex effect* Locus ID† Reference

ﬂbE Transcription factor (putative) Repressor AN0721 Kwon et al. (2010b) fhpA Transcription factor (forkhead) Activator AN4521 Lee et al. (2005) nrdA/msnA Transcription factor (zinc-ﬁnger) Repressor AN1652 Jeon et al. (2009) rcoA Developmental regulation (WD repeat) Activator AN6505 Todd et al. (2006) csnA COP9 signalosome subunit (PCI) Activator AN1491 Busch et al. (2007) csnB COP9 signalosome subunit (PCI) Activator AN4783 Busch et al. (2007) csnD COP9 signalosome subunit (PCI) Bimodal AN1539 Busch et al. (2003, 2007) csnE COP9 signalosome subunit (MPN+ with JAMM Bimodal AN2129 Busch et al. (2007), deneddylase) Nahlik et al. (2010) csnG/acoB COP9 signalosome subunit (PCI) Activator AN3623 Lewis & Champe (1995), Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 Busch et al. (2007) candA-C Protein neddylation Activator AN2458 Helmstaedt et al. (2011) candA-N Protein neddylation Activator AN10306 Helmstaedt et al. (2011)

Endogenous physiological processes ppoA Oxylipin biosynthesis (dioxygenase) Activator AN1967 Tsitsigiannis et al. (2004) ppoB Oxylipin biosynthesis Activator AN6320 Tsitsigiannis et al. (2005) ppoC Oxylipin biosynthesis Repressor AN5028 Tsitsigiannis et al. (2005) noxA Generation of reactive oxygen species (NADPH oxidase) Activator AN5457 Lara-Ortı´z et al. (2003) sidC Intracellular siderophore synthesis (non-ribosomal Activator AN0607 Eisendle et al. (2006) peptide synthetase) trxA Regulation of cellular redox state (thioredoxin system) Activator AN0170 Tho¨ n et al. (2007) mutA a-1,3 Glucanase/mutanase Activator¶ AN7349 Wei et al. (2001) hxtA High-afﬁnity hexose transporter Activator¶ AN6923 Wei et al. (2004)

Ascospore production and maturation grrA Protein ubiquitinylation (F-box) Activator AN10516 Krappmann et al. (2006) samB Cell polarity, nuclear positioning (zinc-ﬁnger) Activator AN0078 Kuger & Fischer (1998) strA Striatin scaffolding and Ca signalling Activator AN8071 Wang et al. (2010) tubB Microtubule assembly (alpha-tubulin) Activator AN0316 Kirk & Morris (1991) vosA Trehalose production Activator AN1959 Ni & Yu (2007)

*Genes listed as ‘Activators’ are required for sexual reproduction, and gene overexpression may enhance sexual fertility. Expression of genes listed as ‘Repressors’ may reduce sexual fertility in general or operate only under particular environmental conditions. See main text for full details. †Comprehensive details of gene and protein sequence are as available from the AspGD website: http://www.aspergillusgenome.org/. Also see list- ing of Aspergillus gene names at: http://www.fgsc.net/Aspergillus/gene_list/loci.html. ‡Acts as a repressor or activator under different environmental conditions and/or at different stages of the sexual cycle. Effects might only be seen under forced gene expression. §Data not yet available. ¶Based on marked upregulation during sexual morphogenesis.

blue light wavelengths, respectively (Blumenstein et al., Perception of environmental signals – light 2005; Bayram et al., 2008a). For example, upregulation of As described above, the sexual cycles of A. nidulans and both veA and nsdA (see below) was seen in a DcryA A. fumigatus are induced only under specific environmen- mutant, suggesting that cryA acts as an upstream repressor tal conditions, and a variety of genes have been identified of these genes (Bayram et al., 2008a). By contrast, LreA linked to sensing of external factors. Regarding photobiol- and LreB activate sexual reproduction in the dark and also ogy, a series of genes that are responsive to light have been promote limited sex in the light (Purschwitz et al., 2008). identified in A. nidulans: the fphA gene encodes a red Furthermore, bimolecular fluorescence assays suggest that light–sensing phytochrome (Blumenstein et al., 2005), the there is physical interaction between lreA, lreB, FphA and lreA and lreB genes encode blue light–sensing white-collar an additional key protein VeA (see below), which together homologues (Purschwitz et al., 2008), and the cryA gene form a nuclear bound ‘light regulator complex’, and it is encodes a blue light- and UVA-sensing cryptochrome the interaction of proteins within this complex that leads (Bayram et al., 2008a). Analysis of A. nidulans deletion to the balance between asexual and sexual development mutants of each of these genes indicates that FphA and under various light/dark conditions (Purschwitz et al., CryA repress sexual reproduction under red and UVA/ 2008; Bayram et al., 2010).

FEMS Microbiol Rev 36 (2012) 165–192 ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 174 P.S. Dyer & C.M. O’Gorman Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021

Fig. 2. Schematic developmental network showing proposed inter-relatedness between genes involved with sexual reproduction in the aspergilli. See main text for details of speciﬁc gene products. Proteins are enclosed in the same box or shaded area when they are believed to act at a similar stage in development, but the particular order of gene activity (or nature of interaction) is not known or has not been experimentally proven. Arrowed blue lines where appropriate indicate activation of following genes, and ﬂat-ended red lines indicate repression. Cleistothecial initials refer to the very earliest stage of cleistothecium development. Most genes illustrated are based on results from the homothallic species Aspergillus nidulans, and it is cautioned that slight differences might be apparent in heterothallic species. Also note that some genes affect multiple points in the sexual cycle so are shown more than once.

A second protein complex known as the ‘velvet’ complex The velvet-like protein VelB shares homology with VeA, also has a pivotal role in light/dark regulation of sexual and the proteins appear to be functionally interdependent development, most likely acting downstream of the previ- with a DvelB gene deletant unable to form cleistothecia ous light regulator complex (Fig. 2). The core components even in the dark; this defect could not be rescued by veA of the velvet complex are three proteins VeA, VelB and overexpression (Bayram et al., 2008b). However, unlike LaeA, together with associated proteins VosA, VipA, VipB veA, velB overexpression did not result in increased num- and VipC (Bayram et al., 2008b, 2010; Bayram & Braus, bers of cleistothecia, so it was suggested that veA deter- 2011). The overall action of the velvet complex in A. nidu- mines the production of the number of cleistothecia lans is to activate sexual development in the dark whilst whereas velB has complementary developmental functions suppressing asexual reproduction. However, analysis of the (Bayram et al., 2008b). In particular, VelB was found to individual protein components has revealed differences in interact with the protein VosA, which is required for pro- their specific activities. The veA gene was first to be studied, duction of trehalose in both asexual conidia and sexual with gene deletion resulting in a complete inability to ascospores, hence affecting the viability of spores (Ni & Yu, reproduce sexually, whereas overexpression resulted in sex- 2007; Bayram et al., 2008b; Sarikaya Bayram et al., 2010). ual morphogenesis under high salt and liquid conditions The VelB–VosA heterodimer was suggested to both repress normally unfavourable for sex (Kim et al., 2002). By con- asexual development and mediate ascospore maturation trast, overexpression of veA in A. fumigatus had no obvious (Sarikaya Bayram et al., 2010). phenotype (Krappmann et al., 2005). Deletion of veA in Meanwhile, it was a major discovery that VeA also inter- A. nidulans also prevented expression of the oxylipin gene acts with the putative methyltransferase LaeA, itself a glo- ppoA (see below) and a regulatory feedback loop has been bal regulator of much secondary metabolism in suggested (Stinnett et al., 2007). Tandem affinity purifica- A. nidulans (Bayram & Braus, 2011). Further analysis into tion studies then identified that VeA interacts with two fur- the role of LaeA in sexual development revealed that LaeA ther proteins VelB and LaeA, with VeA thought to act as a is required for light-mediated inhibition of sex, with dele- putative ‘bridge’ via interactions with the N- and C-termi- tion of laeA resulting in elevated numbers of cleistothecia nal portions of VeA, respectively (Bayram et al., 2008b). being produced both in the light and dark (Sarikaya

Bayram et al., 2010). However, the cleistothecia formed (but not veA) could obviate the density-dependent sup- were signiﬁcantly smaller in size than the wild type. This pression of sclerotial formation both in the light and dark was most likely due to a drastic reduction in the number when using high spore concentrations as the starting of Hu¨lle cells produced, and lack of concomitant mutanase inoculum (Amaike & Keller, 2009). activity, thought to be needed for nursing of the develop- A protein kinase ImeB has also been shown to be ing cleistothecia, that is, LaeA is required for correct Hu¨lle involved in light-mediated inhibition of sexual develop- cell development (Fig. 2). Furthermore, overexpression of ment (Bayram et al., 2009). This is perhaps surprising LaeA in the dark led to a twofold increase in the produc- because the yeast homologue Ime2 is involved speciﬁcally tion of cleistothecia. Thus, LaeA appears to have bimodal in meiosis and the production of ascospores. ImeB deletion activity, suppressing sexual development in the light but strains of A. nidulans produced three- to fourfold

promoting it in the dark. LaeA possibly acts by chemically increased numbers of cleistothecia in the light compared Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 modifying VeA, which was found to have two different cel- to controls and were observed to form Hu¨lle cells in sub- lular isoforms (Sarikaya Bayram et al., 2010; Bayram & merged culture. Expression of the VeA, VelB, stuA and Braus, 2011). Finally, it was observed that VeA also inter- nsdD genes (see below) was increased in the DimeB strain, acted with a nuclear importin protein KapA (Stinnett suggesting that ImeB acts upstream of these genes (Fig. 2). et al., 2007; Bayram et al., 2008b). Taken together, this has Interactions with fphA were also investigated, and it was led to the current model whereby VeA, VelB and KapA are concluded that ImeB and FphA are involved in different thought to be transported to the nucleus in the dark, light-response pathways (Bayram et al., 2009). However, where resulting interaction with LaeA (possibly via an epi- somewhat surprisingly overexpression of imeB led to ‘enor- genetic effect) results in sexual development and altered mous numbers’ of sexual structures independent of any secondary metabolism (Sarikaya Bayram et al., 2010; light effect, so Bayram et al. (2009) suggested that ImeB Bayram & Braus, 2011). This provided evidence of a also had a later role in promoting fruit body formation. molecular link between secondary metabolism and devel- A few final comments on light sensing are that there is opment, with Sarikaya Bayram et al. (2010) speculating possible redundancy in receptor function and crosstalk that these processes might be interconnected, with the between the red light- and blue light–sensing systems production of toxic secondary metabolites of benefit to (Bayram et al., 2010). Also, a VeA homologue has also protect fruiting bodies in the soil environment. shown to be required for sclerotial development in both It is noted that the role of LaeA in sexual development A. parasiticus and A. flavus (Calvo et al., 2004; Duran had not been identified earlier because, as mentioned et al., 2007), and given that these structures are required above, many laboratory strains of A. nidulans contain a for later development of cleistothecia, it can be seen that particular mutation in the veA gene leading to the pro- VeA activity is involved in the co-ordination of sexual duction of a truncated protein lacking the first 37 amino morphogenesis in the aspergilli in general. Furthermore, a acids (Kim et al., 2002). This veA1 mutation leads to a velvet-related VelC protein has recently been identified in lack of red light sensitivity and increased asexual sporula- A. nidulans with activity in enhancing sexual development tion and reduced sexual development in the dark (these in A. nidulans. The velC gene is predicted to encode a 524- being advantageous traits for many laboratory projects). amino acid polypeptide and is one of many potential The mutation also largely eliminates the formation of aer- VosA-interacting proteins identified via yeast two-hybrid ial hyphae, whereas these are formed by wild-type veA (Y2H) screens. Overexpression of velC caused elevated strains that have a velvety appearance, hence the gene numbers of sexual fruiting bodies to be formed, whilst name. And critically it was this N-terminal portion of the deletion of velC resulted in increased asexual spore forma- protein that appears to be required for interaction with tion and decreased fruit body production (Sarikaya LaeA, possibly due to nonfunctionality of a nuclear locali- Bayram et al., 2010; H.S. Park & J.H. Yu, pers. commun.). zation signal in the truncated protein (Stinnett et al., Finally, a mutant screen has identified a series of isolates 2007; Sarikaya Bayram et al., 2010). Unfortunately, this able to undergo sex in the light, but the underlying genetic veA1 effect could cause similarly misleading conclusions basis has been determined for only two of these mutants. to be drawn for other genes impacting on sexual develop- First, deletion of the silA gene (which encodes a zinc-finger ment. LaeA has also been shown to be required for the domain protein) resulted in a blind phenotype showing development of sclerotia in A. flavus, with a complete loss strong induction of cleistothecia in the light (Min et al., of sclerotial formation in a DlaeA strain, whereas an over- 2007; Han et al., 2008c). Second, deletion of the silG gene expressing strain showed a c. sevenfold increase in mass (also encoding a zinc-finger domain protein) similarly of sclerotia in the dark (Kale et al., 2008). There was also resulted in a mutant able to produce high numbers of evidence that laeA negatively regulates veA expression cleistothecia under visible light (Han et al., 2005), that is, (Kale et al., 2008) and that multicopy expression of laeA both SilA and SilG were repressors of sex in the light.

thallic species and are also important in sexual develop- Perception of environmental signals – nutrient mental processes in both heterothallic and homothallic and stress levels species (Debuchy et al., 2010). MAT genes have now been The cross-pathway-control genes cpcA and cpcB are identified from a range of Aspergillus species, including involved in the sensing of amino acid levels (via interaction both known sexual and supposed asexual species (Dyer with a CpcC kinase) and can modulate sexual devel- et al., 2003; Paoletti et al., 2005; Dyer, 2007; Pel et al., opment in A. nidulans. Under limiting amino acid 2007; Ramirez-Prado et al., 2008). conditions, the c-Jun related transcription factor CpcA MAT genes were first described from the homothallic suppresses development beyond the formation of micro- A. nidulans, which was found to contain both alpha- and cleistothecia, even though Hu¨lle cells are present, whereas high mobility group (HMG) classes of MAT genes within b the G subunit protein CpcB represses cpcA expression in the same genome; though, these were unlinked and found Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 the presence of suitable amino acid levels to allow sexual on chromosomes 6 and 3, respectively (Dyer et al., 2003; development (Fig. 2) (Hoffmann et al., 2000). Other Galagan et al., 2005; Paoletti et al., 2007). Functional genes involved in nutrient sensing are the lsdA gene, analysis of these genes, termed MAT1 and MAT2 in rec- which can suppress sex under high salt concentrations ognition of their distinct loci according to standard (Lee et al., 2001), imeB (see above), which has a possible nomenclature for MAT genes, revealed that they were role in inhibiting sex under low glucose conditions essential for correct sexual development, with deletion of although this remains to be proven (Bayram et al., 2009), either gene resulting in the production of significantly phoA, which encodes a cyclin-dependent kinase that is decreased numbers of cleistothecia that were smaller in inhibitory under low phosphorus conditions (Bussink & diameter and entirely sterile (containing no ascospores) Osmani, 1998), and An-pho80, which encodes a cyclin (Paoletti et al., 2007). However, DMAT1 and DMAT2 that conversely promotes sexual development at low strains were able to outcross to each other, that is, the phosphorus concentrations (Wu et al., 2004). A further key observation that manipulation of the MAT genes had esdC gene involved in early sexual development has been lead to heterothallic (self-sterile) strains from a homothal- identified, which encodes a protein containing a glycogen lic parent. The DMAT2 strains also showed occasional binding domain, suggesting a role in regulating develop- prolific production of Hu¨lle cells. Overexpression of both mental processes according to nutrient/metabolic status. MAT1 and MAT2 together resulted in vegetative growth Deletion of esdC led to a total failure to form cleistothe- arrest and development of cleistothecia on media nor- cia even under conditions normally favouring sex, mally unfavourable for sex, further confirming their key although gene overexpression had no obvious effect (Han role in sexual development (Paoletti et al., 2007). A preli- et al., 2008b). Significantly, only very low levels of esdC minary study by Miller et al. (2005) also reported regula- expression were seen in a DveA strain, and reduced levels tion of fruit body formation and meiosis in A. nidulans in a DnsdD strain, suggesting that VeA and NsdD posi- by the unlinked MAT loci. A further preliminary study by tively regulate esdC expression (Fig. 2). Alcocer et al. (2009) reported the presence of a noncod- More generally, various environmental stressors can trig- ing RNA region termed ‘jgaA’ adjacent to MAT2 (matA), ger elevated sexual reproduction. Recently, the addition of whose expression was co-regulated with MAT2 (matA), the nitric oxide (NO)-generating compound diethylenetri- deletion of which led to a 42% decrease in production of amine-NoNoate has been shown to result in an increase in cleistothecia. Given all these findings in the homothallic cleistothecial production and higher expression of the regu- A. nidulans, it is intriguing to speculate whether there latory genes nsdD and steA (see below) (Baidya et al., 2011; might be differential expression of the MAT genes in the Marcos et al., 2011). Linked to this observation, the fhbA nuclei that form the original sexual dikaryon and eventual and fhbB genes encode flavohaemoglobin proteins involved diploid zygote, such that two mating competent nuclei in the reduction and detoxification of NO. Deletion of may come together, one expressing MAT1 and the other fhbA resulted in increased Hu¨lle cell production (Baidya MAT2 to allow nuclear fusion. Indeed, it has been sug- et al., 2011), suggesting that fhbA and fhbB are likely to gested that MAT genes might have a role in determining suppress sexual development under stressful conditions if nuclear identity (Debuchy, 1999; Debuchy et al., 2010) able to mitigate these circumstances. and could be involved in the process of ‘relative hetero- thallism’ in A. nidulans whereby a preference for nuclear outcrossing is evident despite the self-fertile nature of this Genes involved in the mating process species (Pontecorvo, 1953; Hoffmann et al., 2001; Sca- Studies of other filamentous ascomycete species (Pezizo- zzocchio, 2006; Paoletti et al., 2007). mycotina) have established that mating-type (MAT) genes MAT genes were next identified from A. fumigatus, have a key role in determining sexual identity in hetero- which exhibited a standard organization for heterothallic

Pezizomycotina with either a MAT1-1 alpha domain or signiﬁcantly reduced numbers of cleistothecia, whereas a MAT1-2 HMG domain gene being found at a single MAT double deletion strain was completely sterile with no cle- locus in the genome, which showed an ‘idiomorphic’ istothecia produced (although Hu¨lle cells were formed). structure with highly dissimilar regions of DNA sequence These results indicate that pheromone signalling is being present in the MAT locus of the different mating required for fertilization and nuclear karyogamy even in a types, unlike the more normal allelic variants found at a homothalic species, despite the repeated failure to detect gene locus (Paoletti et al., 2005). These genes were later trichogynes or differentiated ascogonia and antheridia in shown to be functional from heterologous expression in A. nidulans (Sohn & Yoon, 2002; Dyer, 2007). Genes for A. nidulans hosts lacking functional MAT1 (MATB) and pheromone precursors (ppgA) and pheromone receptors MAT2 (matA) genes (Grobe & Krappmann, 2008; Pyrzak (preA and preB) have also been described from A. fumiga-

et al., 2008), from mating studies (O’Gorman et al., tus and have been shown to be expressed on solid agar Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 2009), and most recently from gene deletion work where media (Dyer et al., 2003; Paoletti et al., 2005; Szewczyk & DMAT1-1 and DMAT1-2 strains were unable to form fru- Krappmann, 2010). In the first study of their expression, iting bodies (Szewczyk & Krappmann, 2010; Szewczyk there was no perceptible difference in expression of preA et al., 2011). Complementary alpha- and HMG domain and preB between MAT1-1 and MAT1-2 isolates under MAT genes have since also been characterized from Neo- the conditions assayed, although slightly increased ppgA sartorya fischeri (Rydholm et al., 2007) and A. flavus, expression was seen in a MAT1-1 isolate (Paoletti et al., A. parasiticus and P. alliaceus (Ramirez-Prado et al., 2005). Similar results were obtained in a subsequent study 2008). MAT1-1 and MAT1-2 idiomorph regions have in using wild-type isolates, and in addition, a significant addition been identified from A. oryzae, and most decrease in ppgA expression was seen in a DMAT1-1 recently altered gene expression was seen in an A. oryzae strain whereas increased expression was seen in a mating-type replacement strain in which the MAT1-1 DMAT1-2 strain, consistent with ppgA regulation by the gene was substituted by MAT1-2. Many genes, including MAT loci (Szewczyk & Krappmann, 2010). However, the putative a-pheromone precursor gene AoppgA, were despite much bioinformatic effort, no gene encoding a expressed more abundantly in the MAT1-1 strain, and hydrophobic a-type ppgB pheromone has yet been several genes were upregulated in the MAT1-2 strain, detected in the aspergilli. This is most likely due to high suggesting functionality of these mating-type genes sequence divergence amongst such pheromones and the (R. Wada, H. Yamaguchiu, N. Yamamoto, Y. Wagu, relatively short length of product, although potential M. Paoletti, D.B. Archer, J. Maruyama, P.S. Dyer & a-factor efflux genes atrC and atrD, encoding ABC K. Kitamoto, unpublished results). transporters, have been identified from A. nidulans with One key role of MAT genes in heterothallic pezizomy- homologues in A. fumigatus and A. flavus (Andrade et al., cetes is to regulate the production of peptide phero- 2000). Other genes for enzymatic processing and matura- mones, which are involved in the chemotactic attraction tion of pheromones (Saccharomyces cerevisiae ste13, ste14, of mating partners. This involves the production of pher- ste24, kex-1, ram1, ram2 and rce1 homologues) have been omones (from precursor molecules) in a mating-type- detected by BLAST analysis of the A. nidulans genome specific manner and the consequent export of either (Dyer et al., 2003). hydrophilic a-type pheromones (from MAT1-1 isolates) or hydrophobic a-type pheromones (from MAT1-2 iso- Signal transduction pathways lates) (Debuchy et al., 2010). These peptide pheromones are then recognized by cognate G protein-coupled recep- A variety of G protein-coupled signal transduction path- tors (GPCRs), also produced in a mating-type-specific ways have been identified in A. nidulans (Han et al., fashion with a-type pheromones docking to a Pre2 recep- 2004; Galagan et al., 2005). One particular MAP kinase tor (in MAT1-2 isolates) and a-type pheromones docking cascade has been identified as being linked to pheromone to a Pre1 receptor (in MAT1-1 isolates) (Debuchy et al., sensing in A. nidulans, based on homology to the same 2010). Research in the aspergilli first focussed again on transduction pathway in the S. cerevisiae yeast model, and the homothallic A. nidulans, with the identification of a is essential for sexual reproduction to occur (Dyer, 2007). ppgA a-type pheromone and both preA and preB receptor The PreA and PreB receptor proteins are linked to a het- genes arising from genome analysis (Dyer et al., 2003). erotrimeric G protein composed of alpha (a), beta (b) Subsequent experimental work by Seo et al. (2004) and gamma (c) subunits. Deletion of any of the genes revealed that both preA and preB (co-named gprB and encoding these subunits (fadA, sfaD and gpgA, respec- gprA, respectively) were required for normal sexual devel- tively) results in the failure to form cleistothecia, although opment. Deletion of either gene alone resulted in the pro- Hu¨lle cells continued to be formed in abundance under duction of only a few, small cleistothecia containing some conditions (Roseń et al., 1999; Seo et al., 2005),

FEMS Microbiol Rev 36 (2012) 165–192 ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 178 P.S. Dyer & C.M. O’Gorman that is, emphasizing the possible independence of Hu¨lle that RasA has to be predominantly inactivated before cell formation from development of ascogenous hyphae fruit body formation can occur (Hoffmann et al., 2000; and meiosis (see above). The FlbA protein, a regulator of Braus et al., 2002). Meanwhile, Han et al. (2004) identi- G protein signalling because of its GTPase-enhancing fied an extra GPCR encoding gene termed gprD, deletion activity, is also required for the development of both cle- of which resulted in restricted vegetative growth and istothecia and Hu¨lle cells as highlighted by the fact that a production of small colonies densely packed with cleisto- DflbA mutant showed only a fluffy-autolytic phenotype thecia. This implied that GprD-mediated signalling even under conditions favourable for sex (Han et al., negatively regulates sexual development, and indeed nsdD 2001). Deletion of flbA resulted in minimal nsdD expres- transcripts were strongly elevated in a DgprD deletion sion (see below), although no clear effect was seen in strain. GprD is thought to act upstream of GprA/GprB

fadA and sfaD deletion strains (Han et al., 2001); expres- (PreB/PreA) signalling (Seo et al., 2005). A further recep- Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 sion of esdC was also dependent on flbA (Han et al., tor GprK also appears to be essential for the maturation 2008b). A phosducin-like protein PhnA is also required of cleistothecia. A DgprK deletion strain showed accumu- for correct Gbc-mediated signalling, possibly acting as a lation of Hu¨lle cell aggregates but a block in further molecular chaperone. Deletion of phnA resulted in a development of cleistothecia, and gprK transcripts were block in sexual development (although enhanced Hu¨lle highly expressed during sexual development (J.H. Yu, cell formation was noted), and no effect on nsdD expres- pers. commun.). Also related to G protein activity, a sion was observed suggesting a downstream role or a dif- novel Gb-like RACK1 homologue termed GibB has been ferent regulatory branch (Fig. 2) (Seo & Yu, 2006). identified in A. nidulans with similarity to Gib2 in Cryp- A complete MAP kinase cascade in A. nidulans was then tococcus neoformans (Palmer et al., 2006; Q. Kong & J.H. identified downstream of Gbc-mediated signalling, based Yu, pers. commun.). GibB is thought to bind directly to on both genomic and experimental findings (Dyer et al., Gpa1asaGb-like protein to stabilize Gpa1 and facilitate 2003; Galagan et al., 2005; Dyer, 2007) (Fig. 2). It consists its activation and inactivation cycle, and to regulate cAMP of a series of consecutive elements with kinase activity signalling. A gibB deletion mutant produces small colonies [STE20 (MAPKKKK), steC (first identified as steB from a and only c. 2% of the number of cleistothecia compared partial EST sequence) (MAPKKK), STE7 (MAPKK), mpkB to the wild type, and these cleistothecia contain very few (MAPK)] that activate a final homoeodomain protein (if any) ascospores. The cleistothecia of the gibB deletion SteA, which then triggers further stages of sexual develop- mutant are also smaller in size and colourless with abnor- ment. Deletion of individual genes in the pathway has been mal morphology compared to the wild type. This indicates shown to result in sterility, with failure to form cleistothe- that GibB plays an important role in sexual development cia and ascogenous hyphae, although Hu¨lle cells were of A. nidulans, governing a distinct stage of sexual fruiting sometimes produced (Vallim et al., 2000; Wei et al., 2003; (Q. Kong & J.H. Yu, pers. commun.). In addition, a Paoletti et al., 2007). Significantly, Paoletti et al. (2007) homologue of the RIC-8 animal protein, which is crucial found that expression of the whole MAP kinase cascade, for GDP/GTP exchange in the absence of GPCRs or other together with a ste50 kinase regulator, was markedly guanine nucleotide exchange factors, has been identified in upregulated during sexual development. Thus, selfing in A. nidulans.AricA deletion mutant was found to exhibit A. nidulans was interpreted to involve the activation of the extremely restricted hyphal growth and failed to produce same mating processes characteristic of sex in heterothallic any sexual structures (either Hu¨lle cell or cleistothecia) species, that is, self-fertilization does not bypass the even under conditions favourable for sexual development. requirements of outcrossing sex but instead involves These results indicate that RicA functions upstream of the activation of these pathways within a single individual cascade controlling sexual development in A. nidulans (Paoletti et al., 2007). However, unlike heterothallic species, (Fig. 2) (N.J. Kwon & J.H. Yu, pers. commun.). certain aspects of pheromone signalling appeared to be Finally, a putative MAP kinase gene sakA (hogA) has been independent of MAT gene expression, suggesting that at identified as part of a likely transduction pathway sensing least in A. nidulans the MAT genes are primarily required environmental osmotic and oxidative stress (Kawasaki for later stages of sexual development (Paoletti et al., 2007). et al., 2002). Given that DsakA mutants undergo precocious Other signalling cascade components have been identi- sexual development and increased production of cleistothe- fied in A. nidulans in addition to the above pathway ele- cia, it is likely that SakA is a repressor of steA-dependent ments. A second GTP/GDP binding developmental switch morphogenesis under environmentally unfavourable condi- has been reported involving a small G protein encoded tions for sex, but has minimal effect under conducive con- by a ras gene homologue rasA (or A-ras). RasA levels ditions (Kawasaki et al., 2002). SakA is thought to interact modulate asexual sporulation, whilst overexpression of with a further transcription factor AtfA, with deletion rasA results in an acleisothecial phenotype, suggesting of the atfA gene also resulting in derepressed sexual

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 36 (2012) 165–192 Published by Blackwell Publishing Ltd. All rights reserved Sexual development and cryptic sex in the aspergilli 179 development with over a twofold increase in numbers of mutants (Han & Han, 2010). It encodes a zinc-ﬁnger Hu¨lle cells and cleistothecia (Lara-Rojas et al., 2011). GATA-type transcription factor, and DnsdD mutants of A. nidulans failed to produce cleistothecia or Hu¨lle cells under conditions favourable for sex, whereas overexpres- Genes encoding transcription factors and other sion of nsdD led to a dramatic increase in cleistothecia regulatory proteins and fruiting bodies were produced even on high-salt In addition to the genes listed above, a diverse range of media that normally repressed sex (Han et al., 2001). regulatory proteins, many with likely transcription factor Thus, NsdD was concluded to act as a positive activator of activity, have been shown to inﬂuence sexual develop- sex, possibly acting downstream of VeA (Han et al., 2001). ment in the aspergilli (Table 2; Fig. 2). Some of these A Y2H screen later revealed at least two proteins interact-

were first identified owing to their influence on asexual ing with NsdD (Kwon et al., 2003). These interactor of Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 development. For example, the genes stuA and medA with NsdD proteins ‘IndB’ and ‘IndD’ exhibited possible VeA- are best known for their roles in regulating asexual dependent expression (repressed in a veA background, but conidiation. They encode proteins with a basic helix- highly induced in a DveA mutant), suggesting a possible loop-helix (bHLH) region APSES domain and no clearly role in the inhibition of NsdD function possibly due to conserved domain, respectively, However, they are also binding to the zinc-finger region, thereby preventing DNA required for correct sexual development, with medA and binding and transcription by NsdD. An nsdD homologue stuA mutants of A. nidulans failing to produce ascospores has also been identified in A. fumigatus, whose overexpres- and cleistothecia, although medA mutants did produce sion was found to decrease asexual sporulation and induce unorganized masses of Hu¨llle cells (Busby et al., 1996; the production of coiled hyphal structures possibly repre- Wu & Miller, 1997). The devR gene encodes a second senting ascogonia, although no further sexual development bHLH protein, which has also been most intensely char- was observed (Grobe & Krappmann, 2008). A subsequent acterized for its role in asexual conidiation, but is addi- study determined that deletion of the A. fumigatus nsdD tionally essential for sexual development because DdevR gene resulted in loss of crossing ability and also impaired strains entirely failed to form Hu¨lle cells or cleistothecia ability to form heterokaryotic mycelium. Therefore, it was (Tu¨ncher et al., 2004). In parallel, the leucine zipper-like speculated that NsdD might have a role in regulating cell protein DopA is needed for normal asexual sporulation, wall integrity, which could be critical in facilitating hyphal but sexual reproduction was also completely abolished in anastomosis during early mating (Szewczyk & Krapp- a DdopA strain (Pascon & Miller, 2000). In a similar fash- mann, 2010). These findings are consistent with nsdD ion, the WD40 family protein RcoA is required for both mediating early stages of sexual development in homothal- normal asexual development and sexuality, as illustrated lic and heterothallic aspergilli as a whole. by the finding that a DrcoA strain was completely self- A second A. nidulans NSD-related gene, nsdC, has also sterile and unable to form maternal tissues in outcrosses been found to encode a zinc-finger DNA-binding protein,

(Todd et al., 2006). Overexpression of the veA gene in a which features a novel fungal C2HC motif (Kim et al., DrcoA background failed to restore sexual development, 2009). As with NsdD, the NsdC protein also appears to indicating that RcoA acts downstream of VeA in develop- play an important role as a positive regulator of the ini- ment (Fig. 2). Conversely, the zinc-finger transcription tial stages of sexual development and repressor of asexual factor FlbC, which is required for asexual conidiation in sporulation. Deletion of nsdC resulted in the complete A. nidulans, acts mainly as a repressor of sexual develop- loss of fruit body formation and formation of Hu¨lle cells, ment as shown by the abundant formation of Hu¨lle cells whilst overexpression overcame the inhibitory effects of and cleistothecia in a DflbC deletant strain (Kwon et al., certain stressors, allowing sexual development when cul- 2010a). Similarly, the associated regulator of conidiation tures were grown under white light, on high-salt media FlbE also represses sexual development as illustrated again or were subjected to other osmotic stresses (Kim et al., by the increased formation of Hu¨lle cells and cleistothe- 2009). Possible links to VeA and NsdD activity were cia, particularly in a veA1 background, in a DflbE deletant investigated, but it was found that overexpression of nsdC strain (Kwon et al., 2010b). These latter two reports failed to restore sexual development in DveA and DnsdD emphasize the competing nature of asexual and sexual strains and vice versa, indicating that all three genes are reproductive pathways. critical for sex and that they act independently of each Meanwhile, a series of genes have been identified spe- other – there being no obvious change in expression lev- cifically due to their requirement for sexual reproduction. els of nsdC in the DveA and DnsdD strains. One with perhaps the greatest significance for the initial Two further possibly paralogous genes encoding zinc- stages of sexual development is nsdD, so called as it arose finger proteins have been identified from A. nidulans, from a screen of ‘never in sexual development’ (NSD) which also influence sexual development. The first, nosA,

FEMS Microbiol Rev 36 (2012) 165–192 ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 180 P.S. Dyer & C.M. O’Gorman is required for completion of sexual development, with of mature fruit bodies (Nahlik et al., 2010). Most of the DnosA mutants failing to develop beyond sexual primor- CSN effects are assumed to arise from the CsnE deneddy- dia, although very occasional cleistothecia containing lase activity linked to protein turnover (Busch et al., ascospores were formed (Vienken & Fischer, 2006). By 2007), and it has been speculated that the CSN might be contrast, the second, rosA, inhibits sexual development involved with VeA degradation in the light (Braus et al., under certain conditions, with a DrosA mutant able to 2010; Nahlik et al., 2010). Overexpression of veA in a undergo sexual reproduction under low glucose and high DcsnD background failed to overcome the sexual develop- osmolarity conditions that normally inhibit sex (Vienken mental block (Busch et al., 2003). Other CSN subunits et al., 2005). Further studies with various manipulations have also been investigated. A UV mutagenesis screen of the nsdD and nosA genes suggested that NosA most yielded a sterile, aconidial mutant acoB202 (Butnick et al.,

likely acts downstream of NsdD, or in a parallel pathway 1984), and later complementation and gene rescue work Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 (Vienken & Fischer, 2006). This is consistent with the pro- identified a mutation in the acoB gene as responsible for posal of Dyer (2007) that VeA, NsdD, RosA and now the failure to reproduce sexually (Lewis & Champe, NsdC act early in development to control genes inducing 1995). This gene was subsequently found to be part of sexual primordial (Fig. 2), whereas NosA is required for the CSN and was co-named csnG (Busch et al., 2007). the maturation of primordia that already exist, that is, Meanwhile, it was found that deletion of CsnA and CsnB downstream in the developmental pathway. Meanwhile, a also led to identical blocks in development at the primor- forkhead protein transcription factor encoded by the fhpA dia stage as seen in the DcsnD and DcsnE mutants, illus- gene was shown to be required for the production of cleis- trating that formation of the whole CSN complex is tothecia in A. nidulans, although Hu¨lle cell formation was required for correct sexual development (Busch et al., possible in an fhpA disruptant strain (Lee et al., 2005). 2007). In parallel to the CSN investigations, a further An additional major regulatory contributor is the mul- protein Cand1 has recently been described from A. nidu- tiprotein COP9 ‘signalosome’ complex (CSN), which is lans, which also appears to be involved with protein ned- involved with various developmental processes through- dylation. Deletion of N- and C-terminal components, out the eukaryota (Braus et al., 2010). It consists of an encoded by genes candA-N and candA-C, respectively, eight-subunit complex in A. nidulans, and it appears to resulted in a block in sexual development at the initial have contrasting effects on sexual development. Research stage of early nest formation (although Hu¨lle cells were efforts have focussed mainly on two subunits, CsnD and formed), suggesting related developmental functions CsnE (Busch et al., 2003, 2007; Nahlik et al., 2010). It between Cand1 and the CSN (Helmstaedt et al., 2011). was first found that a DcsnD gene deletion mutant was Finally, two transcription factors involved in the forma- blocked in sexual development at the primordial stage tion of sclerotia have been described. First, a bHLH tran- (although Hu¨lle cells were formed as normal), but that scription factor SclR (for sclerotium regulator) has been the mutant was simultaneously ‘blind’ and able to identified from A. oryzae, and deletion of the gene found develop primordia even in the presence of light (Busch to result in sparse sclerotial production whilst overexpres- et al., 2003), that is, CsnD was required not only for cor- sion of sclR led to greater than fivefold production of scle- rect sexual development but also for the suppression of rotia with increased formation of branched aerial hyphae sex in the light. Similarly, the subsequent characterization (Jin et al., 2009, 2011). Second, deletion of a zinc-finger of a DcsnE gene deletion mutant revealed a block in sex- calcineurin-response gene crzA was found to result in the ual development at the primordial stage, but the mutant production of mainly immature sclerotia in A. parasiticus, again demonstrated constitutive formation of primordia that is, a delay in development (Chang, 2008). even in the light. This confirmed that CSN activity is required for sexual morphogenesis but is conversely Genes linked to endogenous physiological involved with light suppression of sex, noting that the processes CsnE subunit appears to have a particular key role in the assembly of the overall CSN complex (Nahlik et al., Various physiological changes occur during the switch 2010). Both of these effects were likely to be mediated from asexual to sexual growth in the aspergilli and the partly via changes in levels of Psi factors regulating sporu- subsequent development of cleistothecia and accessory tis- lation (see below) as increased ppoA gene expression and sues. Some of these changes have been well documented altered hormone balance were observed in the DcsnE at a biochemical level, and over the past decade, some of mutant. Also, reduced expression of cell wall–degrading the genes involved in these processes have been identified. enzymes was seen in the DcsnE strain, with the suggestion For example, a chemically related group of oxylipin hor- that normal CSN activity is required for the release of mones termed ‘Psi’ factors (for precocious sexual induc- materials needed for wall remodelling and development ers), derived from linoleic acid and related to mammalian

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 36 (2012) 165–192 Published by Blackwell Publishing Ltd. All rights reserved Sexual development and cryptic sex in the aspergilli 181 prostaglandin hormones, has been identified. These Ppo function has also been investigated in A. flavus include the members psiBa [8-hydroxy-(9,12)-octadecadi- and A. fumigatus.InA. flavus, homologues to the enoic acid (8-HODE)] and psiCa (5,8-diHODE), which A. nidulans ppoA, ppoB and ppoC genes were discovered can trigger a switch from asexual to sexual growth, or by genome BLAST analysis, together with an additional psiAa (similar structure to psiCa but with a lactone ring ppoD dioxygenase homologue (Brown et al., 2009). In replacing the hydroxyl group at the 5′ position), which A. flavus, oxylipin products appear to play a vital role in can repress sex and induce asexual reproduction (Champe mediating spore concentration–dependent quorum sens- et al., 1994; Tsitsigiannis et al., 2004). And three oxylipin ing, which influences production of sclerotia (as described biosynthetic genes, ppoA, ppoB and ppoC (mnemonic for above). Deletion of an Aflox gene, encoding a lipoxygen- psi factor–producing oxygenase), encoding linoleate diol ase (LOX), was found to minimize the density-dependent

synthases were subsequently identified from A. nidulans inhibition of sclerotial production at high spore concen- Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 (Tsitsigiannis et al., 2004, 2005; Brodhun & Feussner, trations, suggesting that a LOX-derived metabolite is 2011). PpoA has been shown to catalyse the formation of responsible for the activation of asexual sporulation and various 8′ oxylipins including psiBa and pisCa (Tsitsi- the simultaneous repression of sex under these conditions giannis et al., 2004; Brodhun & Feussner, 2011). PpoC is (Brown et al., 2008). Downregulation of the four genes mainly involved in the formation of 10′ products (e.g. ppoA, ppoB, ppoC and Aflox in a DppoD background 10-HPODE) and appears not to form Psi factors directly, resulted in a similar outcome, with a dramatic increase in but has an indirect role in the production pathway possi- sclerotial production (up to 500-fold) at high spore con- bly leading to psiA formation (Brodhun & Feussner, centrations (Brown et al., 2009). It was suggested that 2011). Thus, ppoA and ppoC have antagonistic roles. Sex- ppoC and Aflox were particularly inhibitory to sclerotial ual and asexual development are characterized by differ- formation, whilst ppoD promoted sclerotial production, ent ratios between 8-hydroxy-(9)-octadecanoic acid and possibly via production of an opposing oxylipin that psiB, the ratio shifting from 1 : 8 in darkness (sexual) to functioned at low spore concentration. As noted above, 1 : 3 in light (asexual) (Bayram & Braus, 2011). The bio- multicopy expression of laeA was able to abolish this chemical properties of PpoB remain unclear (Brodhun & quorum-like phenomenon (Amaike & Keller, 2009). In Feussner, 2011). Consistent with these findings, deletion the case of A. fumigatus, deletion of only ppoC led to a of ppoA resulted in a major reduction in ascospore pro- distinct phenotype, with altered condium size, germina- duction, but an increase in asexual sporulation, whilst tion and stress tolerance (Dagenais et al., 2008). ppoA overexpression led to increased sexual reproduction Another physiological signal for early sexual morpho- (Tsitsigiannis et al., 2004). Interestingly, virtually no ppoA genesis appears to be the cellular oxidation state. Deletion expression was detected in a DveA strain and increased of the noxA gene in A. nidulans, which encodes an expression was seen in csnD mutant strains, suggesting NADPH oxidase able to produce reactive oxygen species, that VeA and CsnD may regulate ppoA expression resulted in a block in cleistiothecial development at the (Fig. 2) (Tsitsigiannis et al., 2004; Dyer, 2007). Deletion initial stage (although Hu¨lle cells were present) (Lara-Ortı´z of ppoB resulted in decreased sex linked to downregula- et al., 2003). It was proposed that noxA might contribute tion of ppoA but upregulation of ppoC, that is, suggesting to a hyperoxidant state that triggers cell proliferation, a regulatory loop between the three genes (Fig. 2). By apoptosis and cell wall developmental changes during early contrast, deletion of ppoC led to an increase in produc- sexual morphogenesis. NoxA expression was independent tion of cleistothecia, demonstrating the opposing activity of SteA and StuA activity but suppressed by SakA (Fig. 2). of ppoB and ppoC (Tsitsigiannis et al., 2005; Brown et al., A related catalase–peroxidase gene cpeA has also been 2009). Surprisingly, a triple DppoADppoBDppoC mutant identified that possibly offers protection against oxidative exhibited increased activation of sexual development with damage (Scherer et al., 2002). Linked to these observa- the production of Hu¨lle cells and even some cleistothecia tions, two protein components TrxA and TrxR forming a under water, correlated with greatly increased VeA classic cytoplasmic thioredoxin system have been identified expression (Tsitsigiannis et al., 2005). Thus, although in A. nidulans. Deletion of trxA resulted in failure to form oxylipins have an important role in the induction of sex, any sexual structures, although this could be alleviated by other parameters can independently trigger morphogene- addition of low levels of reduced glutathione, again sis (Dyer, 2007). In the context of asexual and sexual indicating that cellular redox state has a vital role in reproduction in A. nidulans, it is intriguing to speculate allowing sexual development to occur (Tho¨n et al., 2007). whether the fluG gene, suggested to regulate asexual coni- Possibly also linked to cellular redox state, Eisendle et al. diation via production of a mysterious extracellular devel- (2006) found that deletion of the sidC gene, encoding a opmental signal (Lee & Adams, 1994), might mediate its nonribosomal peptide synthetase required for production effect partly through an influence on psiA synthesis. of an intracellular siderophore, led to impaired asexual

FEMS Microbiol Rev 36 (2012) 165–192 ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 182 P.S. Dyer & C.M. O’Gorman and sexual development even in iron-replete conditions. duced defective, small cleistothecia containing only very Cleistothecia failed to develop even though Hu¨lle cells few viable semi-transparent ascospores (Ni & Yu, 2007). were formed, suggesting that iron homoeostasis is vital for A further gene with a role in ascosporogenesis is the cellular developmental processes. Also linked to cellular striatin homologue strA, which encodes a putative multi- redox state, a homologue of the yeast MSN2 transcription domain scaffolding protein with a possible role in cal- factor involved in stress response has been identiﬁed in the cium signalling in the endomembrane system (Wang aspergilli. The NrdA (originally termed MsnA but renamed et al., 2010). Deletion of strA resulted in the production for being a negative regulator of differentiation) protein of abnormally small cleistothecia with various defects in appears to play a role in the maintenance of cellular oxida- ascospore formation, for example failure to develop asci, tive state, although with contrasting roles in different failure of ascospores to separate correctly in asci, and

aspergilli. Deletion of nrdA led to increased production of production of abnormally shaped ascospores. However, in Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 cleistothecia in A. nidulans, whereas deletion of the msnA a VeA (although not VeA1) background, the majority of homologue led to loss of sclerotial production in A. flavus ascospores developed normally and were viable, a good and A. parasiticus, so subtle differences are apparent (Jeon example of a ‘leaky’ mutation ensuring ascospore produc- et al., 2009; Chang et al., 2010). tion as a possible survival mechanism despite mutational Sexual morphogenesis in A. nidulans has also been damage (Dyer, 2007). Overexpression of strA resulted in a shown to be correlated with increased a-glucanase activ- c. twofold increase in cleistothecial production under sex- ity, possibly linked to energy release for the development inducing conditions, increased production of protocleis- of cleistothecia (Zonneveld, 1977). Genes encoding an tothecia under repressive high-salt conditions, and the a-1,3 glucanase/mutanase (mutA) and high-affinity hex- formation of Hu¨llle cells even in shaking liquid culture ose transporter (hxtA), which both show elevated levels of (Wang et al., 2010). This illustrates the positive role of expression during sexual development, have subsequently StrA in sexual development and shows that it promotes been identified (Wei et al., 2001, 2004). However, no other parts of the sexual cycle in addition to ascospore obvious inhibition of sexual development was seen in formation (Fig. 2). mutA and hxtA deletion strains, suggesting redundancy in A series of other mutants of A. nidulans have been pro- carbon usage and uptake systems. duced by UV mutagenesis with various defects in recombination ability and ascospore development, for example no croziers, arrest at prekaryogamy or meiotic prophase Later developmental stages – ascospore or metaphase, nondelineation of ascospores, and colour- production less or blue ascospores. However, the genes involved are Certain genes appear to be required for the later stages of as yet unknown although some have been localized to the sexual cycle in A. nidulans, being linked specifically to certain chromosomes (Apirion, 1963; Osman et al., 1993; the production and maturation of ascospores. For exam- Swart et al., 2001). It has been estimated that 50–100 ple, a deletion mutant of the tubB gene, encoding an genes might be involved specifically in ascospore forma- alpha-tubulin protein, exhibited normal development and tion based on complementation analysis (Swart et al., formed asci, but these contained only a single nuclear 2001) with the term mei proposed for certain of these mass consistent with a block in karyogamy and/or meosis sexual sporulation mutants (Bruggeman et al., 2003a). It following zygote formation (Kirk & Morris, 1991). Simi- is noted that BLAST searching has detected at least 100 larly, a deletion mutant of the grrA gene, encoding a fun- genes in A. nidulans with homologues to genes in bud- gal F-box protein (likely involved with protein ding and fission yeasts known to be involved in karyoga- ubiquitinylation and degradation/recycling), was able to my and meiosis (Galagan et al., 2005). form Hu¨lle cells and cleistothecia, but was blocked at the point of meiosis and ascospore formation with only empty Insights into cryptic sexuality in the asci observed. This particular F-box protein appears to be aspergilli specifically required for ascosporogenesis, as evidenced by upregulation of its transcript during cleistothecial develop- In the final section of this review, recent findings ment (Krappmann et al., 2006). Deletion of the samB concerning sexual development in the once-presumed gene, encoding a zinc-finger protein associated with ‘asexual’ species A. fumigatus, A. flavus, A. parasiticus and nuclear positioning, also resulted in failure to produce via- A. nominus will be described and implications for cryptic ble ascospores and led to spore lysis (Kuger & Fischer, sexuality in the aspergilli discussed. 1998). And as mentioned above, the vosA gene is required The majority of Aspergillus species are only known for the production of trehalose in sexual ascospores, as to reproduce by asexual means. As discussed in the illustrated by the fact that a DvosA mutant strain pro- Introduction, this is very surprising given the many

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 36 (2012) 165–192 Published by Blackwell Publishing Ltd. All rights reserved Sexual development and cryptic sex in the aspergilli 183 perceived benefits of reproducing by both sexual and 2009). The species also causes aspergillosis in other mam- asexual means, as opposed to obligate asexuality. How- mals and birds (Tell, 2005). ever, there is now accumulating evidence that many sup- The importance of A. fumigatus to human health led posedly ‘asexual’ Aspergillus species might in fact have the to a clinical isolate (Af293) being the first Aspergillus gen- potential to reproduce by sexual means (Horn et al., ome to be publically sequenced. BLAST analysis revealed 2009a, b, c, 2011; O’Gorman et al., 2009) and thus that, despite its asexual status, the Af293 genome pos- exhibit ‘cryptic sexuality’ or ‘covert’ sexual reproduction, sessed a complete set of known genes linked to sexual with a so far hidden sexual state that has yet to be dis- reproduction, 215 in total, that is, a full suite of ‘sexual covered (Ku¨ck & Po¨ggeler, 2009; Heitman, 2010). This is machinery’ (Galagan et al., 2005). Importantly, all of highly significant given that many of the asexual aspergilli these sex-related genes appeared functional, with no

are of either medical or industrial importance. Thus, the mutations within any of their coding regions. The same Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 presence of a sexual cycle would impact on the popula- suite of genes was found when a second A. fumigatus iso- tion biology of a species, perhaps enabling more rapid late (A1163) was sequenced, providing further evidence evolution of antifungal resistance in pathogens, whilst for a species-wide sexual potential (Fedorova et al., 2008). simultaneously providing a valuable genetic tool for Characteristic conserved a-domain (MAT1-1) and HMG classical genetic analyses and for strain improvement by domain (MAT1-2) genes were present at a MAT locus in industry. the A1163 and Af293 isolates, respectively (Po¨ggeler, Evidence for cryptic/covert sexuality has come from 2002; Galagan et al., 2005; Fedorova et al., 2008), consis- data drawn from diverse research areas such as popula- tent with experimental cloning of a MAT1-1 idiomorph tion biology analyses, genome analysis of sex-related (Paoletti et al., 2005), in the typical arrangement for het- genes, the distribution of complementary mating types in erothallic (outcrossing) species. When a worldwide collec- nature, expression of sex-related genes and classical labo- tion of 290 clinical and environmental isolates of ratory mating studies (Dyer & Paoletti, 2005; Heitman, A. fumigatus was screened, MAT1-1 and MAT1-2 mating 2010). Such studies have had a great impact, leading to types were found in near-equal proportion, suggesting the discovery of sexual states in the following Aspergillus that sexual reproduction was still occurring frequently species, which were once considered purely asexual. enough to maintain the balance of mating types (Paoletti et al., 2005). In addition, the pheromone precursor (ppgA), pheromone receptor (preA and preB) and mating- Aspergillus fumigatus type genes (MAT1-1 and MAT1-2) were all shown to be Aspergillus fumigatus is one of the most ubiquitous fungi expressed during normal vegetative growth (Paoletti worldwide, colonizing diverse habitats but being typically et al., 2005). found in soil and organic debris such as compost. It is This information together with evidence of gene well known for the proliﬁc production of asexual conidia recombination from population genetic studies (Varga & (Samson et al., 2009). Inhalation of these spores by To´th, 2003; Paoletti et al., 2005; Pringle et al., 2005; Bain immunocompetent individuals rarely has any adverse et al., 2007) and the presence of close taxonomic relatives effect because of efﬁcient elimination by the innate with sexual states (Samson et al., 2009) suggested the immune system (Latge´, 1999). However, since the early presence of cryptic or ‘clandestine’ sexuality in A. fumiga- 1990s, A. fumigatus has become the most prevalent air- tus, which was suggested to be ‘holding back the truth borne fungal pathogen worldwide. This has been due to about its sexuality’ (Gow, 2005). Critically, the necessary the increase in numbers of immunocompromised conditions for a sexual cycle remained unclear. However, patients, whose weakened immune systems allow it to O’Gorman et al. (2009) then made a major breakthrough cause opportunistic infections (Latge´, 1999; Dagenais & when they were able to induce a fully functional sexual Keller, 2009). Groups at risk include those undergoing cycle in vitro and show that it leads to the production of chemotherapeutic regimes for bone marrow and organ recombinant ascospore progeny; the teleomorph was transplantation and patients with HIV/AIDS. Even with named N. fumigata (Fig. 3). A key underpinning to this treatment, mortality rates of 50–80% are reported (Lin work was the availability of known MAT1-1 and MAT1-2 et al., 2001; Richardson & Lass-Flo¨rl, 2008). Manifesta- isolates, as revealed by the PCR diagnostic of Paoletti tions of disease range from saprophytic growth in et al. (2005), meaning that directed crosses could be set pre-existing cavities (aspergillomas) to life-threatening up between isolates with potential sexual compatibility. invasive aspergillosis. In addition, the airborne conidia This was previously not possible, and therefore crossing of A. fumigatus can trigger hypersensitivity diseases efforts might otherwise have been confounded because of including asthma, allergic sinusitis and allergic broncho- the abortive presence of isolates of the same mating type. pulmonary aspergillosis (Latge´, 1999; Dagenais & Keller, The required conditions for sexual reproduction were

(a) (b)

Fig. 3. Scanning electron micrographs of a cleistothecium and ascospores of Neosartorya fumigata, the teleomorph of Aspergillus fumigatus. (a) Single cleistothecium showing wall composed of a network of layers of interlocking hyphae, characteristic of the genus Neosartorya; scale bar indicates 100 lm (O’Gorman et al., 2009). (b) Two ascospores

exhibiting two equatorial crests and ridged Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 ornamentation; scale bar indicates 2 lm (O’Gorman et al., 2009). growth on oatmeal agar at 30 °C in the dark with of aflatoxin contamination allowable in these foods (Van restricted aeration for up to 6 months (O’Gorman et al., Edmond & Jonker, 2005). In addition to being mycotoxi- 2009). Significantly, the authors have since successfully genic, A. flavus is also an opportunistic human pathogen, mated combinations of both clinical and environmental being the second most common agent of aspergillosis isolates from a wide variety of locations other than the after A. fumigatus (Krishnan et al., 2009). Irish isolates used in their original study, confirming that Both species are characterized by very high levels of fertility is neither limited to the environmental strains genetic diversity, as seen by the large number of vegeta- nor is geographically restricted to Ireland (C.M. O’Gor- tive compatibility groups (VCG) and DNA fingerprint man, S. Swilaiman, J. Sugui, K.J. Kwon-Chung & P.S. types that have been found (Horn et al., 1996; Horn, Dyer, unpublished results). Szewczyk & Krappmann 2007). This considerable diversity could not be attributed (2010) also recently reported the successful crossing of to asexual reproduction alone, suggesting that either mei- two unrelated clinical strains of A. fumigatus, one of otic or parasexual recombination was responsible. Indeed, which was the genome-sequenced isolate Af293 (Galagan related population genetic studies have revealed that et al., 2005). A. flavus and A. parasiticus both have recombining popula- The discovery of a sexual cycle in A. fumigatus was tion structures (Geiser et al., 1998; Carbone et al., 2007). highly significant as it helped to explain many puzzling A sample population of A. flavus from Australia showed aspects of its biology and evolution. These ‘puzzles’ evidence of recombination, with two reproductively iso- included the presence of numerous different genotypes lated clades found (Geiser et al., 1998), whilst analysis of within populations and the successful defence of the gen- a field collection of A. parasiticus from Georgia in the ome against repetitive elements (Latge´, 1999). Using clas- USA found evidence of both a history of recombination sical genetic analyses, it will now be possible to gauge the and contemporary recombination from phylogenetic net- contribution of meiotic recombination to the ongoing work analysis of the aflatoxin gene cluster (Carbone et al., evolution of A. fumigatus. 2007). Genome analysis also revealed that both species have an idiomorphic MAT locus arrangement characteristic of the heterothallic Pezizomycotina, with a single biall- Aspergillus flavus and Aspergillus parasiticus elic MAT locus (Ramirez-Prado et al., 2008). In addition, Aspergillus flavus and A. parasiticus are two very closely the MAT genes were shown to be expressed in the two related members of Aspergillus section Flavi. Morphologi- species, and there were near-equal numbers of isolates of cally, the two species are near identical, but they can be each mating type present in close proximity in the Geor- easily separated by mycotoxin profiling, AFLP fingerprint- gia field populations. Thus, both species appeared to ing and DNA sequencing (Horn et al., 1996; Montiel exhibit cryptic sexuality. This was re-enforced by genome et al., 2003; Peterson, 2008). They are the major world- screening that has revealed a full complement of appar- wide producers of aflatoxins, one of the most potent ently functional genes required for sexual reproduction in groups of natural toxins known to man with carcinogenic A. flavus (C.E. Eagle & P.S. Dyer, unpublished results). and acute immunosuppressive and neurotoxic effects Following the discovery of sexual reproduction in (John, 2008). Aflatoxins contaminate a wide variety of A. fumigatus (O’Gorman et al., 2009), similar break- agricultural crops including cereals, oilseeds and nuts, throughs were then published shortly afterwards concern- and over 100 countries have strict limits on the amount ing A. parasiticus and A. flavus. The sexual cycles of both

ª 2011 Federation of European Microbiological Societies FEMS Microbiol Rev 36 (2012) 165–192 Published by Blackwell Publishing Ltd. All rights reserved Sexual development and cryptic sex in the aspergilli 185 species were induced using the same method: strains of 5–10 months. The discovery of the teleomorph was con- opposite mating type were paired on mixed cereal agar in sistent with earlier findings of cryptic sexual recombina- darkness at 30 °C in sealed plastic bags for 6–9 months tion based on patterns of gene polymorphisms (Peterson (A. parasiticus)or6–11 months (A. flavus) (Horn et al., et al., 2001). However, A. nomius differed from the other 2009a, c). Hardened sclerotia were produced during this species in that overall fertility of the field isolates studied period, with the sexual ascocarps found to develop within was markedly lower than A. parasiticus and A. flavus, their matrices. Sclerotial production had previously been ascospores only being produced in 24% of crosses. Even observed in A. parasiticus and A. flavus and was postu- in such fertile crosses, in most cases, only 1–5% of sclero- lated to function as a survival mechanism for the fungi tia contained asci with ascospores (Horn et al., 2011). A during adverse environmental conditions. However, here similar lack of sexual fertility has been described in vari-

they acted as a repository for the cleistothecia. It was pre- ous other pezizomycete species (Dyer et al., 1992; Dyer & Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 viously hypothesized that the sclerotia of A. flavus would Paoletti, 2005). A variety of factors might contribute to be capable of harbouring fruiting bodies given the right this apparent low fertility. For example, the crossing conditions (Rai et al., 1967; Geiser et al., 1998). The two conditions used in the laboratory may not have been sexual stages were assigned to the genus Petromyces, the optimal, leading to decreased fertility. Alternatively, dif- teleomorphic genus associated with Aspergillus section ferences in VCG between isolates may have influenced Flavi (Horn et al., 2009a, b, c). Previously, the homothal- mating success (Dyer et al., 1992). In the case of A. nomi- lic Aspergillus alliaceus was the only other known sexual us, the VCGs of the paired isolates were unknown, unlike species within this section (Raper & Fennell, 1965). the situation for the A. flavus and A. parasiticus crosses Morphologically, the ascospores of the three Petromyces (Horn & Greene, 1995; Horn et al., 2011). Finally, it has species are extremely similar; Petromyces flavus and been suggested that an overall ‘slow decline’ in sexual Petromyces parasiticus are near indistinguishable, whilst reproduction and fertility within populations may be P. alliaceus differs by having slightly smaller ascospores linked to evolution towards asexuality, because of factors that are smooth instead of finely roughened (Horn et al., such as decreased or suppressed expression of sex-related 2009a, b, c). Significantly, many species in Aspergillus genes (Dyer & Paoletti, 2005). section Flavi produce sclerotia, suggesting that sexual Aspergillus nomius also exhibited one other unusual reproduction may also be possible in these species. feature in that whilst most field isolates were of either It is not surprising that the crossing conditions for MAT1-1 or MAT1-2 genotype, some occasional isolates A. flavus and A. parasiticus are very similar to the condi- were of joint MAT1-1/MAT1-2 genotype. These were not tions required for A. fumigatus mating as they are all self-fertile, but were able to outcross to both MAT1-1 and soil saprotrophs (Klich, 2002a). Ascospores are typically MAT1-2 isolates. Such unusual ‘dual maters’ have been highly resistant structures, with a thick cell wall that con- reported elsewhere in the Pezizomycotina albeit very fers thermotolerance (Baggerman & Samson, 1988; Di- rarely (Debuchy et al., 2010; Amselem et al., 2011). The jksterhuis, 2007). Their long incubation periods for fruit exact genetic organisation of this genotype remains to be body development may have evolved as a mechanism to determined. outlast unfavourable environmental conditions. As most other known sexual Aspergillus species complete their sex- Concluding remarks ual cycles within several weeks, this may explain why sexual reproduction in these three species remained From the above, it can be seen that discoveries concern- unobserved for so long. ing the sexual biology of the aspergilli have made a major contribution to the understanding of fungal sex in general. Numerous genes involved in the initiation and co- Aspergillus nomius and beyond ordination of sexual reproduction have already been Most recently, a sexual cycle has also been induced in the described from the aspergilli (Table 2), and many parts of previously ‘asexual’ aflatoxin producing species A. nomius the developmental pathway proposed in Fig. 2 might be (Horn et al., 2011). This is also a member of the section applicable to the Pezizomycotina as a whole. The avail- Flavi, and similar protocols were used to induce the sex- ability of considerable genome resources and an ongoing ual stage as were used for A. parasiticus and A. flavus, project to systematically create a series of knockout con- that is, identification of compatible mating types using a structs for all genes of A. nidulans (http://www.fgsc.net/ PCR diagnostic, and then crossing used a mixed spore Aspergillus/ko_cassettes/cassetteannouncement.htm) pro- inoculum on mixed cereal agar. As seen with A. parasiti- vides wonderful opportunities to identify further aspects cus and A. flavus, the cleistothecia of A. nomius were of the sexual circuitry of the group. Indeed, it has been formed within sclerotia over a long time period of estimated that up to 2000 genes might be involved in

FEMS Microbiol Rev 36 (2012) 165–192 ª 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 186 P.S. Dyer & C.M. O’Gorman developmental and differentiation processes (Braus et al., References 2002), so many more genes remain to be characterized. For example, classical genetic studies have identified a Agnihotri VP (1968) Effects of nitrogenous compounds on sclerotium formation in Aspergillus niger. Can J Microbiol series of mutants ranging from acleistothecial to dense 14: 1253–1258. cleistothecial forms (sgp, aco, acl, lcl, dcl) whose molecular Agnihotri VP (1969) Some nutritional and environmental genetic basis remains unknown (Houghton, 1970; Zonne- factors affecting growth and production of sclerotia by a veld, 1974; Scherer & Fischer, 1998). Genomic technolo- strain of Aspergillus niger. Can J Microbiol 15: 835–840. gies such as EST and microarray analysis and whole Alcocer JG, Miller K & Miller B (2009) Mating type genes transcriptome shotgun sequencing (‘RNA seq’) offer fur- control self/non-self recognition during fertilization, ther opportunities to identify genes differentially regu- karyogamy and meiosis in the filamentous fungi. Fungal 56 lated during sexual morphogenesis. Already some results Genet Rep (suppl): 138. Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 are available from pilot studies of cleistothecial develop- Amaike S & Keller NP (2009) Distinct roles for VeA and LaeA ment in A. nidulans (Jeong et al., 2000, 2001) and sclero- in development and pathogenesis of Aspergillus flavus. tial development in A. flavus (Cary et al., 2007). In Eukaryot Cell 8: 1051–1060. addition, there is promise that bioinformatic analyses will Amselem J, Cuomo C, van Kan J et al. (2011) Genomic yield novel insights into development. For example, a analysis of the necrotrophic fungal pathogens Sclerotinia 7 reciprocal smallest distance analysis was recently used to sclerotiorum and Botrytis cinerea. PLoS Genet : e1002230. identify 245 orthologous genes shared between the sclero- Andrade AC, Van Nistelrooy JGM, Peery RB, Skatrud PL & DeWaard MA (2000) The role of ABC transporters from tial-forming species Botrytis cinerea, Sclerotinia sclerotio- Aspergillus nidulans in protection against cytotoxic agents rum, A. flavus and A. oryzae, but absent from the and in antibiotic production. Mol Gen Genet 263: 966–977. nonsclerotial-producing A. nidulans and A. fumigatus; the Apirion D (1963) Formal and physiological genetics of ascospore characterization of such genes offers a potential foothold colour in Aspergillus nidulans. Genet Res 4: 276–283. into a broader understanding of sclerotial development Archer DB & Dyer PS (2004) From genomics to post- and evolution (Amselem et al., 2011). genomics in Aspergillus. Curr Opin Microbiol 7: 499–504. Meanwhile a ‘sexual revolution’ is ongoing in the sup- Baggerman WI & Samson RA (1988) Heat resistance of fungal posedly asexual aspergilli (Horn et al., 2009a, c, 2011; spores. Introduction to Food-Borne Fungi, 3rd edn (Samson O’Gorman et al., 2009). The discoveries of sexual cycles RA & van Reenen-Hoekstra ES, eds), pp. 262–267. in more ‘asexual’ species will further serve to validate the Centraalbureau voor Schimmelcultures, Utrecht. joining of mitosporic fungi with their meiosporic rela- Baidya S, Cary JW, Grayburn WS & Calvo AM (2011) Role of tives and perhaps would only come as a surprise to the nitric oxide and flavohemoglobin homologous genes in ‘laboratory voyeur’ rather than a population geneticist Aspergillus nidulans sexual development and mycotoxin 77 – (Gow, 2005). Attempts are currently underway to induce production. Appl Environ Microbiol : 5524 5528. the sexual stages of various other asexual aspergilli of Bain JM, Tavanti A, Davidson AD, Jacobsen MD, Shaw D, Gow NAR & Odds FC (2007) Multilocus sequence typing of industrial and medical importance (S. Swilaiman, C.M. the pathogenic fungus Aspergillus fumigatus. J Clin Microbiol O’Gorman, H. Darbyshir & P.S. Dyer, unpublished data). 45: 1469–1477. It is cautioned that the sexual cycles of some species Bayram O¨ & Braus GH (2011) Coordination of secondary might still elude discovery because of the fastidious nat- metabolism and development in fungi: the velvet family of ure of their sexual demands (Kwon-Chung & Sugui, regulatory proteins. FEMS Microbiol Rev, in press, doi: 2009) or genuine evolution to asexuality. Also, species 10.1111/j.1574-6976.2011.00285.x. should arguably not be viewed as either sexual or asexual Bayram O¨ , Biesemann C, Krappmann S, Galland P & Braus but rather as being composed of isolates present on a GH (2008a) More than a repair enzyme: Aspergillus nidulans continuum of sexual fertility (Dyer & Paoletti, 2005). photolyase-like CryA is a regulator of sexual development. However, the availability of MAT diagnostic tools now Mol Biol Cell 19: 3254–3262. allows targeted crosses to be set up between compatible Bayram O¨ , Krappmann S, Ni M et al. (2008b) VelB/VeA/LaeA MAT1-1 and MAT1-2 isolates of asexual species offering complex coordinates light signal with fungal development 320 – the exciting prospect of further sexual revelations in the and secondary metabolism. Science : 1504 1506. ¨ near future. Bayram O, Sari F, Braus GH & Irniger S (2009) The protein kinase ImeB is required for light-mediated inhibition of sexual development and for mycotoxin production in Acknowledgements Aspergillus nidulans. Mol Microbiol 71: 1278–1295. Bayram O¨ , Braus GH, Fisher R & Rodriguez-Romero J (2010) We thank the Wellcome Trust (UK) for research funding, Spotlight on Aspergillus nidulans photosensory system. and Jae-Hyuk Yu and Ana Calvo for sharing unpublished Fungal Genet Biol 47: 900–908. results.

Benjamin CR (1955) Ascocarps of Aspergillus and Penicillium. an intact JAMM motif is required for fungal fruit body Mycologia 47: 669–687. formation. P Natl Acad Sci USA 104: 8089–8094. Bennett JW (2009) Aspergillus: a primer for the novice. Med Bussink HJ & Osmani SA (1998) A cyclin-dependent kinase Mycol 47(suppl): S1–S8. family member (PHOA) is required to link developmental Bennett JW (2010) An overview of the genus Aspergillus. fate to environmental conditions in Aspergillus nidulans. Aspergillus Molecular Biology and Genomics (Machida M & EMBO J 17: 3990–4003. Gomi K, eds), pp. 1–17. Caister Academic Press, Norfolk. Butnick NZ, Tager NL, Kurtz MB & Champe SP (1984) Bennett JW, Fernholz FA & Lee LS (1978) Effect of light on Genetic analysis of mutants of Aspergillus nidulans blocked aﬂatoxins, anthraquinones, and sclerotia in Aspergillus ﬂavus at an early stage of sporulation. J Bacteriol 160: 541–545. and Aspergillus parasiticus. Mycologia 70: 104–116. Calvo AM, Hinze LL, Gardner HW & Keller NP (1999) Blaser P (1975) Taxonomic and physiological studies on the Sporogenic effect of polyunsaturated fatty acids on

genus Eurotium. Sydowia 28:1–49. development of Aspergillus spp. Appl Environ Microbiol 65: Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 Blumenstein A, Vienken K, Tasler R, Purschwitz J, Veith D, 3668–3673. Frankenberg-Dinkel N & Fischer R (2005) The Aspergillus Calvo AM, Bok J, Brooks W & Keller NP (2004) VeA is nidulans phytochrome FphA represses sexual development required for toxin and sclerotial production in Aspergillus in red light. Curr Biol 15: 1833–1838. parasticus. Appl Environ Microbiol 70: 4733–4739. Braus GH, Krappman S & Eckert SE (2002) Sexual Carbone I, Jakobek JL, Ramirez-Prado JH & Horn BW (2007) development in ascomycetes. Fruit body formation of Recombination, balancing selection and adaptive evolution Aspergillus nidulans. Molecular Biology of Fungal in the aflatoxin gene cluster of Aspergillus parasiticus. Mol Development (Osiewacz HD, ed), pp. 215–244. Marcel Ecol 16: 4401–4417. Dekker, New York. Carvalho MDF, Baracho MS & Baracho IR (2002) Braus GH, Irniger S & Bayram O¨ (2010) Fungal development Investigation of the nuclei of Hu¨lle cells of Aspergillus and the COP9 signalosome. Curr Opin Microbiol 13: 672– nidulans. Genet Mol Biol 25: 485–488. 676. Cary HW, O’Brian GR, Nielsen DM, Nierman W, Harris- Brodhun F & Feussner I (2011) Oxylipins in fungi. FEBS J Coward P, Yu J, Bhatnagar D, Cleveland TE, Payne GA & 278: 1047–1063. Calvo AM (2007) Elucidation of veA-dependent genes Brown SH, Zarnowsji R, Sharpee WC & Keller NP (2008) associated with aflatoxin and sclerotial production in Morphological transition governed by density dependence Aspergillus flavus by functional genomics. Appl Microbiol and lipoxygenase activity in Aspergillus flavus. Appl Environ Biotechnol 76: 1107–1118. Microbiol 74: 5674–5685. Champe SP, Nagle DL & Yager LN (1994) Sexual sporulation. Brown SH, Scott JB, Bhaheetharan J, Sharpee WC, Milde L, Aspergillus: 50 Years On (Martinelli SD & Kinghorn JR, Wilson RA & Keller NP (2009) Oxygenase coordination is eds), pp. 429–454. Elsevier, Amsterdam. required for morphological transition and the host–fungus Chang PK (2008) Aspergillus parasiticus crzA, which encodes interaction of Aspergillus flavus. Mol Plant Microbe Interact calcineurin response zinc-finger protein, is required for 22 : 882–894. aflatoxin production under calcium stress. Int J Mol Sci 9: Bruggeman J, Debets AJM, Swart K & Hoekstra RF (2003a) 2027–2043. Male and female crosses of Aspergillus nidulans as revealed Chang PK, Scharfenstein LL, Luo M, Mahoney N, Molyneux by vegetatively incompatible parents. Fungal Genet Biol 39: RJ, Yu J, Brown RL & Campbell BC (2010) Loss of msnA,a 136–141. putative stress regulatory gene, in Aspergillus parasiticus and Bruggeman J, Debets AJM, Wijngaarden PJ, Arjan J, DeVisser Aspergillus flavus increased production of conidia, aflatoxins GM & Hoekstra RF (2003b) Sex slows down the and kojic acid. Toxins 3:82–104. accumulation of deleterious mutations in the homothallic Dagenais TRT & Keller NP (2009) Pathogenesis of Aspergillus fungus Aspergillus nidulans. Genetics 164: 479–485. fumigatus in invasive aspergillosis. Clin Microbiol Rev 22: Bruggeman J, Debets AJM & Hoekstra RF (2004) Selection 447–465. arena in Aspergillus nidulans. Fungal Genet Biol 41: 181–188. Dagenais TRT, Chung D, Giles SS, Hull CM, Andes D & Busby TM, Miller KY & Miller BL (1996) Suppression and Keller NP (2008) Defects in condiophore development enhancement of the Aspergillus nidulans medusa mutation and conidium–macrophage interaction in a dioxygenase by altered dosage of the bristle and stunted genes. Genetics mutant of Aspergillus fumigatus. Infect Immun 76: 3214– 143: 155–163. 3220. Busch S, Eckert SE, Krappmann S & Braus GH (2003) The Debuchy R (1999) Internuclear recognition: a possible COP9 signalosome is an essential regulator of development connection between euascomycetes and in the filamentous fungus Aspergillus nidulans. Mol homobasidiomycetes. Fungal Genet Biol 27 : 218–223. Microbiol 49: 717–730. Debuchy R, Berteaux-Lecellier V & Silar P (2010) Mating Busch S, Schwier EU, Nahlik K, Bayram O¨ , Helmstaedt K, systems and sexual morphogenesis in ascomycetes. Cellular Draht OW, Krappmann S, Valerius O, Lipscomb WN & and Molecular Biology of Filamentous Fungi (Borkovich KA & Braus GH (2007) An eight-subunit COP9 signalosome with Ebbole DJ, eds), pp. 501–535. ASM Press, Washington, DC.

Dijksterhuis J (2007) Heat-resistant ascospores. Food Han DM, Han YJ, Chae KS, Jahng KY & Lee YH (1994) Mycology: A Multifaceted Approach to Fungi and Food Effect of various carbon sources on the development of (Dijksterhuis J & Samson RA, eds), pp. 101–117. CRC Aspergillus nidulans with velA+ or velA1 allele. Kor J Genet Press, Boca Raton, FL. 22: 332–337. Duran RM, Cary JW & Calvo AM (2007) Production of Han KH, Han KY, Yu JH, Chae KS, Jahng KY & Han DM cyclopiazonic acid, aflatrem, and aflatoxin by Aspergillus (2001) The nsdD gene encodes a putative GATA-type flavus is regulated by veA, a gene necessary for sclerotial transcription factor necessary for sexual development of formation. Appl Environ Microbiol 73: 1158–1168. Aspergillus nidulans. Mol Microbiol 41: 299–309. Dyer PS (2007) Sexual reproduction and significance of MAT in Han KH, Lee DB, Kim JH, Kim MS, Han KY, Kim WS, Park the aspergilli. Sex in Fungi: Molecular Determination and YS, Kim HB & Han DM (2003) Environmental factors Evolutionary Principles (Heitman J, Kronstad JW, Taylor JW & affecting development of Aspergillus nidulans. J Microbiol 41:

Casselton LA, eds), pp. 123–142. ASM Press, Washington, DC. 34–40. Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 Dyer PS & Paoletti M (2005) Reproduction in Aspergillus Han KH, Seo JA & Yu JH (2004) A putative G protein- fumigatus: sexuality in a supposedly asexual species? Med coupled receptor negatively controls sexual reproduction in Mycol 43(suppl): S7–S14. Aspergillus nidulans. Mol Microbiol 51: 1333–1345. Dyer PS, Ingram DS & Johnstone K (1992) The control of Han KH, Lee BY & Han DM (2005) The Aspergillus nidulans sexual morphogenesis in the Ascomycotina. Biol Rev 67: silG gene functions in repression of sexual development in 421–458. response to light. Fungal Genet Newsl 52(suppl): 57. Dyer PS, Ingram DS & Johnstone K (1993) Evidence for the Han DM, Chae KS & Han KH (2008a) Sexual development in involvement of linoleic acid and other endogenous lipid Aspergillus nidulans. The Aspergilli Genomics, Medical factors in perithecial development of Nectria haematococca Aspects, Biotechnology, and Research Methods (Gouldman mating population VI. Mycol Res 97: 485–496. GH & Osmani SA, eds), pp. 279–299. CRC Press, Boca Dyer PS, Paoletti M & Archer DB (2003) Genomics reveals Raton, FL. sexual secrets of Aspergillus. Microbiology 149: 2301–2303. Han KH, Kim JH, Moon H, Kim S, Lee SS, Han DM, Jahng Eckert SE, Hoffmann B, Wanke C & Braus GH (1999) Sexual KY & Chae KS (2008b) The Aspergillus nidulans esdC (early development of Aspergillus nidulans in auxotrophic strains. sexual development) gene is necessary for sexual Arch Microbiol 172: 157–166. development and is controlled by veA and a heterotrimeric Eisendle M, Schrettl M, Kragl C, Mu¨ller D, Illmer P & Haas H G protein. Fungal Genet Biol 45: 310–318. (2006) The intracellular siderophore ferricrocin is involved Han SY, Ko JA, Kim JH, Han KY, Han KH & Han DM in iron storage, oxidative-stress resistance, germination and (2008c) Isolation and functional analysis of the silA gene sexual devlopment in Aspergillus nidulans. Eukaryot Cell 5: that controls sexual development in response to light in 1596–1603. Aspergillus nidulans. Kor J Mycol 36: 189–195. Fedorova ND, Khaldi N, Joardar VS et al. (2008) Genomic Hawksworth DL, Crous PW, Redhead SA et al. (2011) The islands in the pathogenic filamentous fungus Aspergillus Amsterdam declaration on fungal nomenclature. IMA fumigatus. PLoS Genet 4: e1000046. Fungus 2: 105–112. Galagan JE, Calvo SE, Cuomo C et al. (2005) Sequencing of Heath LAF & Eggins HOW (1965) Effects of light, Aspergillus nidulans and comparative analysis with temperature and nutrients on the production of conidia A. fumigatus and A. oryzae. Nature 438: 1105–1115. and sclerotia by forms of Aspergillus japonicus. Experientia Geiser DM (2008) Sexual structures in Aspergillus: 21: 385–386. morphology, importance and genomics. Med Mycol 47 Heitman J (2010) Evolution of eukaryotic microbial pathogens (suppl): S21–S26. via covert sexual reproduction. Cell Host Microbe 8:86–99. Geiser DM, Pitt JI & Taylor JW (1998) Cryptic speciation and Helmstaedt K, Schwier EU, Christmann M, Nahlik K, recombination in the aflatoxin producing fungus Aspergillus Westermann M, Harting R, Grond S, Busch S & Braus GH flavus. P Natl Acad Sci USA 95: 388–393. (2011) Recruitment of the inhibitor Cand1 to the cullin Gow NAR (2005) Fungal genomics: forensic evidence of sexual substrate adaptor site mediates interaction to the activity. Curr Biol 15: R509–R511. neddylation site. Mol Biol Cell 22: 153–164. Grobe V & Krappmann S (2008) The asexual pathogen Hoffmann B, Wanke C, Kirsten SA, Paglia K & Braus GH Aspergillus fumigatus expresses functional determinants of (2000) c-Jun and RACK1 homologues regulate a control Aspergillus nidulans sexual development. Eukaryot Cell 7: point for sexual development in Aspergillus nidulans. Mol 1724–1732. Microbiol 37:28–41. Han KH (2009) Molecular genetics of Emericella nidulans Hoffmann B, Eckert SE, Krappman S & Braus GH (2001) sexual development. Microbiology 37: 171–182. Sexual diploids of Aspergillus nidulans do not form by Han KH & Han DM (2010) Genetics and genomics of sexual random fusion of nuclei in the heterokaryon. Genetics 157: development of Aspergillus nidulans. Aspergillus Molecular 141–147. Biology and Genomics (Machida M & Gomi K, eds), Horn BW (2007) Biodiversity of Aspergillus section Flavi in the pp. 115–137. Caister Academic Press, Norfolk. United States: a review. Food Addit Contam 24: 1088–1101.

Horn BW & Greene RL (1995) Vegetative compatibility within Kim HR, Chae KS, Han KH & Han DM (2009) The nsdC gene

populations of Aspergillus flavus, A. parasiticus, and encoding a putative C2H2-type transcription factor is a key A. tamarii from a peanut field. Mycologia 87: 324–332. activator of sexual development in Aspergillus nidulans. Horn BW, Greene RL, Sobolev VS, Dorner JW, Powell JH & Genetics 182: 771–783. Layton RC (1996) Association of morphology and Kirk KE & Morris NR (1991) The tubB alpha-tubulin gene is mycotoxin production with vegetative compatibility groups essential for sexual development in Aspergillus nidulans. in Aspergillus flavus, A. parasiticus, and A. tamarii. Mycologia Genes Dev 5: 2014–2023. 88: 574–587. Klich MA (2002a) Identification of Common Aspergillus Species. Horn BW, Ramirez-Prado JH & Carbone I (2009a) Sexual Centraalbureau voor Schimmelcultures, Utrecht. reproduction and recombination in the aflatoxin-producing Klich MA (2002b) Biogeography of Aspergillus species in soil fungus Aspergillus parasiticus. Fungal Genet Biol 46: 169–175. and litter. Mycologia 94:21–27.

Horn BW, Ramirez-Prado JH & Carbone I (2009b) The sexual Krappmann S, Bayram O¨ & Braus GH (2005) Deletion and Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 state of Aspergillus parasiticus. Mycologia 101: 275–280. exchange of the Aspergillus fumigatus veA locus via a Horn BW, Moore GG & Carbone I (2009c) Sexual novel recyclable marker module. Eukaryot Cell 4: 1298– reproduction in Aspergillus flavus. Mycologia 101: 423–429. 1307. Horn BW, Moore GG & Carbone I (2011) Sexual Krappmann S, Jung N, Medic B, Prade RA & Braus GH reproduction in aflatoxin-producing Aspergillus nomius. (2006) The Aspergillus nidulans F-box protein GrrA links Mycologia 103: 174–183. SCF activity to meiosis. Mol Microbiol 61:76–88. Houghton JA (1970) A new class of slow-growing non-perithecial Krishnan S, Manavathu EK & Candrasekar PH (2009) mutants of Aspergillus nidulans. Genet Res 16: 285–292. Aspergillus flavus: an emerging non-fumigatus Aspergillus Jeon MH, Hagiwara D, Yoshimi A, Abe K, Han DM & Chae species of significance. Mycoses 52: 206–222. SK (2009) Analysis of nrdA, a negative regulator of Ku¨ck U & Po¨ggeler S (2009) Cryptic sex in fungi. Fungal Biol differentiation in Aspergillus nidulans. Fungal Genet Rep Rev 23:86–90. 56(suppl): 138. Kuger M & Fischer R (1998) Integrity of a Zn finger-like Jeong HY, Han DM, Jahng KY & Chae KS (2000) The rpl16a domain in SamB is crucial for morphogenesis in gene for ribosomal protein L16A identified from expressed ascomycetous fungi. EMBO J 17: 204–214. sequence tags is differentially expressed during sexual Kwon NJ, Han DM & Chae KS (2003) veA-dependent development of Aspergillus nidulans. Fungal Genet Biol 31: expression of IndB and indD encoding proteins that interact 69–78. with NSDD, a GATA-type transcription factor required for Jeong HY, Cho GB, Han KY, Kim J, Han DM, Jahng KY & sexual development in Aspergillus nidulans. Fungal Genet Chae KS (2001) Differential expression of house-keeping Newsl 50(suppl): 70. genes of Aspergillus nidulans during sexual development. Kwon NJ, Garzia A, Espeso EA, Ugalde U & Yu JH (2010a)

Gene 262: 215–219. FlbC is a putative nuclear C2H2 transcription factor Jin FJ, Takahashi T, Machida M & Koyama Y (2009) regulating development in Aspergillus nidulans. Mol Identification of a basic helix-loop-helix-type transcription Microbiol 77: 1203–1219. regulator gene in Aspergillus oryzae by systematically Kwon NJ, Shin KS & Yu JH (2010b) Characterization of the deleting large chromosomal segments. Appl Environ developmental regulator FlbE in Aspergillus fumigatus and Microbiol 75: 5943–5951. Aspergillus nidulans. Fungal Genet Biol 47: 981–993. Jin FJ, Takahashi T, Matsushima KI, Hara S, Shinohara Y, Kwon-Chung KJ & Sugui JA (2009) Sexual reproduction in Maruyama JI, Kitamoto K & Koyama Y (2011) SclR, a basic Aspergillus species of medical or economic importance: why helix-loop-helix transcription factor, regulates hyphal so fastidious? Trends Microbiol 17: 481–487. morphology and promotes sclerotial formation in Aspergillus Lara-Ortı´z T, Rosas-Riveros H & Aguirre J (2003) Reactive oryzae. Eukaryot Cell 10: 945–955. oxygen species generated by microbial NADPH oxidase John L (2008) Discovery of aflatoxins and significant historical NoxA regulate sexual development in Aspergillus nidulans. features. Toxin Rev 27: 171–201. Mol Microbiol 50: 1241–1255. Kale SP, Milde L, Trapp MK, Frisvad JC, Keller NP & Bok JW Lara-Rojas F, Sańchez O, Kawasaki L & Aguirre J (2011) (2008) Requirement of LaeA for secondary metabolism and Aspergillus nidulans transcription factor AtfA interacts with sclerotial production in Aspergillus flavus. Fungal Genet Biol the MAPK SakA to regulate general stress responses, 45: 1422–1429. development and spore functions. Mol Microbiol 80: 436– Kawasaki L, Sańchez O, Shiozaki K & Aguirre J (2002) SakA 454. MAP kinase is involved in stress signal transduction, sexual Latge´ JP (1999) Aspergillus fumigatus and aspergillosis. Clin development and spore viability in Aspergillus nidulans. Mol Microbiol Rev 12: 310–350. Microbiol 45: 1153–1163. Lee BN & Adams TH (1994) The Aspergillus nidulans fluG Kim HS, Han KY, Kim KJ, Han DM, Jahng KY & Chae KS gene is required for production of an extracellular (2002) The veA gene activates sexual development in developmental signal and is related to prokaryotic glutamine Aspergillus nidulans. Fungal Genet Biol 37:72–80. synthetase. Genes Dev 8: 641–651.

Lee DW, Kim S, Kim SJ, Han DM, Jahng KY & Chae KS Penicillium and Aspergillus. Integration of Modern Taxonomic (2001) The lsdA gene is necessary for sexual development Methods for Penicllium and Aspergillus Classiﬁcation inhibition by a salt in Aspergillus nidulans. Curr Genet 39: (Samson RA & Pitt JI, eds), pp. 83–99. Harwood Academic 237–243. Publishers, Amsterdam. Lee BY, Han SY, Choi HG, Kim JH, Han KH & Han DM Osman F, Tomsett B & Strike P (1993) The isolation of (2005) Screening of growth- or developmental-related genes mutagen-sensitive nuv mutants of Aspergillus nidulans and by using genomic library with inducible promoter in their effects on mitotic recombination. Genetics 134: 445– Aspergillus nidulans. J MIcrobiol 43: 523–528. 454. Lewis C & Champe SP (1995) A pre-induction sporulation Palmer DA, Thompson JK, Li L, Prat A & Wang P (2006) gene from Aspergillus nidulans. Microbiology 141: 1821–1828. Gib2, a novel Gbeta-like/RACK1 homolog, functions as a Lin SJ, Schranz J & Teutsch SM (2001) Aspergillosis case– Gbeta subunit in cAMP signaling and is essential in

fatality rate: systematic review of the literature. Clin Infect Cryptococcus neoformans. J Biol Chem 281: 32596–32605. Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 Dis 32: 358–366. Paoletti M, Rydholm C, Schwier EU, Anderson MJ, Szakacs G, Malloch D & Cain RF (1972) The trichomataceae: ascomycetes Lutzoni F, Debeaupuis JP, Latge´ JP, Denning DW & Dyer with Aspergillus, Paecilomyces, and Penicillium imperfect PS (2005) Evidence for sexuality in the opportunistic fungal stages. Can J Bot 50: 2613–2628. pathogen Aspergillus fumigatus. Curr Biol 15: 1242–1248. Marcos AT, Schinko T, Strauss J & Cańovas D (2011) Nitric Paoletti M, Seymour FA, Alcocer MJC, Kaur N, Calvo AM, oxide (NO) is a morphogenetic signal in fungi. Fungal Archer DB & Dyer PS (2007) Mating type and the genetic Genet Rep 58(suppl): 171. basis of self-fertility in the model fungus Aspergillus McAlpin CE (2004) Synnema and sclerotium production in nidulans. Curr Biol 17: 1384–1389. Aspergillus caelatus and the influence of substrate Pascon RC & Miller BL (2000) Morphogenesis in Aspergillus composition on their development in selected strains. nidulans requires Dopey (DopA), a member of a novel Mycologia 96: 937–947. family of leucine zipper-like proteins conserved from yeast McAlpin CE & Wicklow DT (2005) Culture media and sources to humans. Mol Microbiol 36: 1250–1264. of nitrogen promoting the formation of stromata and Paster N & Chet I (1980) Effects of environmental factors on ascocarps in Petromyces alliaceus (Aspergillus section Flavi). growth and sclerotium formation in Aspergillus ochraceus. Can J Bot 51: 765–771. Can J Bot 58: 1844–1850. Miller KY, Nowell A & Miller BL (2005) Differential regulation Pel HJ, de Winde JH, Archer DB et al. (2007) Genome of fruitbody development and meiosis by the unlinked sequencing and analysis of the versatile cell factory Aspergillus nidulans mating type loci. Fungal Genet Newsl 52 Aspergillus niger CBS 513.88. Nat Biotechnol 25: 221–231. (suppl): 184. Peterson SW (2008) Phylogenetic analysis of Aspergillus species Min JY, Kim HR, Han KH & Han DM (2007) Isolation and using DNA sequences from four loci. Mycologia 100: 205– characterization of Aspergillus nidulans mutants which 226. undergo sexual development in light exposure. Kor J Peterson SW, Ito Y, Horn BW & Goto T (2001) Aspergillus Microbiol 43:77–82. bombycis, a new aflatoxigenic species and genetic variation Montiel D, Dickinson MJ, Lee HA, Dyer PS, Jeenes DJ, in its sibling species, A. nomius. Mycologia 93: 689–703. Roberts IN, James S, Fuller LJ, Matsuchima K & Archer DB Po¨ggeler S (2002) Genomic evidence for mating abilities in the (2003) Genetic differentiation of the Aspergillus section Flavi asexual pathogen Aspergillus fumigatus. Curr Genet 42: 153– complex using AFLP fingerprints. Mycol Res 107: 1427– 160. 1434. Po¨ggeler S, Nowrousian M & Ku¨ck U (2006) Fruiting-body Nahlik K, Dumkow M, Bayram O¨ et al. (2010) The COP9 development in ascomycetes. The Mycota I, Growth, signalosome mediates transcriptional and metabolic Differentiation and Sexuality, 2nd edn (Kuës U & Fischer R, response to hormones, oxidative stress protection and cell eds), pp. 325–355. Springer-Verlag, Berlin. wall rearrangement during fungal development. Mol Polacheck I & Rosenberger RF (1977) Aspergillus nidulans Microbiol 78: 964–979. mutant lacking alpha-(1,3)-glucan, melanin, and Nesci A, Morales M & Etcheverry M (2007) Interrelation of cleistothecia. J Bacteriol 132: 650–656. growth media and water activity in sclerotia characteristics Pontecorvo G (1953) The genetics of Aspergillus nidulans. of Aspergillus section Flavi. Lett Appl Microbiol 44: 149–154. Adv Genet 5: 141–238. Ni M & Yu JH (2007) A novel regulator couples sporogenesis Pringle A, Baker DM, Platt JL, Wares JP, Latge JP & Taylor and trehalose biogenesis in Aspergillus nidulans. PLoS ONE JW (2005) Cryptic speciation in the cosmopolitan and 2: e970. clonal human pathogenic fungus Aspergillus fumigatus. O’Gorman CM, Fuller HT & Dyer PS (2009) Discovery of a Evolution 59: 1886–1899. sexual cycle in the opportunistic fungal pathogen Aspergillus Purschwitz J, Mu¨ller S, Kastner C, Scho¨ser M, Haas H, Espeso fumigatus. Nature 457: 471–474. EA, Atoui A, Calvo AM & Fisher R (2008) Functional and Okuda T, Klich MA, Seifert KA & Ando K (2000) Media and physical interaction of the blue- and red-light sensors in incubation effects on morphological characteristics of Aspergillus nidulans. Curr Biol 18: 255–259.

Pyrzak W, Miller KY & Miller BL (2008) The mating type Schoustra S, Ryndle HD, Dali R & Kassen R (2010) Fitness- protein Mat1-2 from asexual Aspergillus fumigatus drives associated sexual reproduction in a ﬁlamentous fungus. sexual reproduction in fertile Aspergillus nidulans. Eukaryot Curr Biol 20: 1350–1355. Cell 7: 1029–1040. Seo JA & Yu JH (2006) The phosducin-like protein PhnA is Rai JN, Tewari JP & Sinha AK (1967) Effect of environmental required for Gbc-mediated signaling for vegetative growth, conditions on sclerotia and cleistothecia production in developmental control, and toxin biosynthesis in Aspergillus Aspergillus. Mycopathol Mycol Appl 31: 209–224. nidulans. Eukaryot Cell 5: 400–410. Ramirez-Prado JH, Moore GG, Horn BW & Carbone I (2008) Seo JA, Han KH & Yu JH (2004) The gprA and gprB genes Characterization and population analysis of the mating-type encode putative G protein-coupled receptors required for genes in Aspergillus ﬂavus and A. parasiticus. Fungal Genet self-fertilization in Aspergillus nidulans. Mol Microbiol 53: Biol 45: 1292–1299. 1611–1623.

Raper JR (1966) Genetics of Sexuality in Higher Fungi. Ronald Seo JA, Han KH & Yu JH (2005) Multiple roles of a Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 Press, New York. heterotrimeric G-protein c-subunit in governing growth and Raper KB & Fennell DI (1965) The Genus Aspergillus. The development of Aspergillus nidulans. Genetics 171:81–89. Williams & Wilkins Company, Baltimore, MD. Sohn KT & Yoon KS (2002) Ultrastructural study on the Rice WR (2002) Evolution of sex: experimental tests of the cleistothecium development in Aspergillus nidulans. adaptive significance of sexual reproduction. Nat Rev Genet Mycobiology 30: 117–127. 3: 241–251. Stinnett SM, Espero EA, Cobenõ L, Arau´ jo-Bazań L & Calvo Richardson M & Lass-Flo¨rl C (2008) Changing epidemiology AM (2007) Aspergillus VeA subcellular localization is of systemic fungal infections. Clin Microbiol Infect 14: dependent on the importin a carrier and on light. Mol 5–24. Microbiol 63: 242–255. Roseń S, Yu JH & Adams TH (1999) The Aspergillus nidulans Swart K, Van Heemst DV, Slakhorst M, Debets F & Heyting C sfaD gene encodes a G protein b subunit that is required for (2001) Isolation and characterization of sexual sporulation normal growth and repression of sporulation. EMBO J 18: mutants of Aspergillus nidulans. Fungal Genet Biol 33:25–35. 5592–5600. Szewczyk E & Krappmann S (2010) Conserved regulators of Rydholm C, Dyer PS & Lutzoni F (2007) DNA sequence mating are essential for Aspergillus fumigatus cleistothecium characterization and molecular evolution of MAT1 and formation. Eukaryot Cell 9: 774–783. MAT2 mating-type loci of the self-compatible ascomycete Szewczyk E, Schro¨ter F & Krappmann S (2011) Back to the mold Neosartorya fischeri. Eukaryot Cell 6: 868–874. future – sexuality of the human pathogen Aspergillus Samson RA & Varga J (2010) Molecular systematics of fumigatus. Fungal Genet Rep 58(suppl): 213. Aspergillus and its teleomorphs. Aspergillus Molecular Tao L, Gao N, Chen S & Yu JH (2010) The choC gene Biology and Genomics (Machida M & Gomi K, eds), encoding a putative phospholipid methyltransferase is pp. 19–40. Caister Academic Press, Norfolk. essential for growth and development in Aspergillus Samson RA, Houbraken JAMP, Kuijpers AFA, Frank JM & nidulans. Curr Genet 56: 283–296. Frisvad JC (2004) New ochratoxin A or sclerotium Taylor JW, Jacobson DJ & Fisher MC (1999) The evolution of producing species in Aspergillus section Nigri. Stud Mycol asexual fungi: reproduction, speciation and classification. 50:45–61. Annu Rev Phytopathol 37: 197–246. Samson RA, Varga J & Dyer PS (2009) Morphology and Tell LA (2005) Aspergillosis in mammals and birds: impact on reproductive mode of Aspergillus fumigatus. Aspergillus veterinary medicine. Med Mycol 43(suppl): S71–S73. fumigatus and Aspergillosis (Latge´ JP & Steinbach WJ, eds), Thakur ML (1973) Effect of osmotic concentration and pH on pp. 7–13. ASM Press, Washington, DC. sclerotia and cleistothecia production in alkaline and fertile Sarikaya Bayram O¨ , Bayram O¨ , Valerius O, Park HS, Irniger S, soil Aspergilli. Microbios 7: 215–220. Gerke J, Ni M, Han K-H, Yu J-H & Braus GH (2010) LaeA Tho¨n M, Al-Abdallah Q, Hortschansky P & Brakhageb AA control of velvet family regulatory proteins for light- (2007) The thioredoxin system of the filamentous fungus dependent development and fungal cell-type specificity. Aspergillus nidulans: impact on development and oxidative PLoS Genet 6: e1001226. stress response. J Biol Chem 282: 27259–27269. Scazzocchio C (2006) Aspergillus genomes: secret sex and the Todd RB, Hynes MJ & Andrianopoulos A (2006) The secrets of sex. Trends Genet 22: 521–525. Aspergillus nidulans rcoA gene is required for veA-dependent Scherer M & Fischer R (1998) Purification and sexual development. Genetics 174: 1685–1688. characterization of laccase II of Aspergillus nidulans. Arch Todd RB, Davis MA & Hynes MJ (2007) Genetic manipulation Microbiol 170:78–84. of Aspergillus nidulans: meiotic progeny for genetic analysis Scherer M, Wei H, Liese R & Fischer R (2002) Aspergillus and strain construction. Nat Protoc 2: 811–821. nidulans catalase-peroxidase gene (cpeA)is Tsitsigiannis DI, Zarnowski R & Keller NP (2004) The lipid transcriptionally induced during sexual development body protein, PpoA, coordinates sexual and asexual through the transcription factor StuA. Eukaryot Cell 1: sporulation in Aspergillus nidulans. J Biol Chem 279: 11344– 725–735. 11353.

Tsitsigiannis DI, Kowieski TM, Zarnowski R & Keller NP of Aspergillus nidulans is induced in vegetative hyphae upon (2005) Three putative oxylipin biosynthetic genes integrate starvation and in ascogenous hyphae during cleistothecium sexual and asexual development in Aspergillus nidulans. development. Fungal Genet Biol 41: 148–156. Microbiology 151: 1809–1821. Wright S & Finnegan D (2001) Genome evolution: sex and the Tu¨ncher A, Reinke H, Martic G, Caruso ML & Brakhage AA transposable element. Curr Biol 11: R296–R299. (2004) A basic-region helix-loop-helix protein-encoding Wu J & Miller BL (1997) Aspergillus asexual reproduction gene (devR) involved in the development of Aspergillus and sexual reproduction are differentially affected by nidulans. Mol Microbiol 52: 227–241. transcriptional and translational mechanisms Vallim MA, Miller KY & Miller BL (2000) Aspergillus SteA regulated stunted gene expression. Mol Cell Biol 17: 6191– +2 (sterile12-like) is a homeodomain-C2/H2-Zn ﬁnger 6201. transcription factor required for sexual reproduction. Mol Wu D, Dou X, Hashmi SB & Osmani SA (2004) The Pho80-

Microbiol 36: 290–301. like cyclin of Aspergillus nidulans regulates development Downloaded from https://academic.oup.com/femsre/article/36/1/165/534783 by guest on 24 September 2021 Van Edmond HP & Jonker MA (2005) Worldwide regulations independently of its role in phosphate acquisition. J Biol on aﬂatoxins. Aﬂatoxin and Food Safety (Abbas HK, ed), Chem 279: 37693–37703. pp. 77–93. Taylor & Francis, Boca Raton, FL. Yager LN (1992) Early developmental events during asexual Varga J & To´th B (2003) Genetic variability and reproductive and sexual sporulation in Aspergillus nidulans. Aspergillus: mode of Aspergillus fumigatus. Infect Genet Evol 3:3–17. Biological and Industrial Applications (Bennett JW & Klich

Vienken K & Fischer R (2006) The Zn(II)2Cys6 putative MA, eds), pp. 19–42. Butterworth, Boston, MA. transcription factor NosA controls fruiting body formation Zonneveld BJ (1972) Morphogenesis in Aspergillus nidulans: in Aspergillus nidulans. Mol Microbiol 61: 544–554. the signiﬁcance of alpha-1,3-glucan synthesis of the cell wall

Vienken K, Scherer M & Fischer R (2005) The Zn(II)2Cys6 and alpha-1,3-glucanase for cleistothecium development. putative Aspergillus nidulans transcription factor repressor of Biochim Biophys Acta 273: 174–187. sexual development inhibits sexual development under low- Zonneveld BJM (1974) a-1,3-Glucan synthesis correlated with carbon conditions and in submersed culture. Genetics 169: a-1,3 glucanase synthesis, conidiation and fructiﬁcation in 619–630. morphogenetic mutants of Aspergillus nidulans. J Gen Wang CL, Shim WB & Shaw BD (2010) Aspergillus nidulans Microbiol 81: 445–451. striatin (Stra) mediates sexual development and localizes to Zonneveld BJM (1975) Sexual differentiation in Aspergillus the endoplasmic reticulum. Fungal Genet Biol 47: 789–799. nidulans: the requirement for manganese and its effect on Wei H, Scherer KM, Singh A, Liese R & Fischer R (2001) a-1,3 glucan synthesis and degradation. Arch Microbiol 105: Aspergillus nidulans a-1,3 glucanase (mutanase), mutA,is 101–104. expressed during sexual development and mobilizes mutan. Zonneveld BJM (1977) Biochemistry and ultrastructure of Fungal Genet Biol 34: 217–227. sexual development in Aspergillus. Genetics and Physiology of Wei H, Requena N & Fischer R (2003) The MAPKK kinase Aspergillus (Smith JE & Pateman JA, eds), pp. 59–80. SteC regulates conidiophore morphology and is essential for Academic Press, London. heterokaryon formation and sexual development in the Zonneveld BJM (1988a) Morphology of initials and number of homothallic fungus Aspergillus nidulans. Mol Microbiol 47: nuclei initiating cleistothecia in Aspergillus nidulans. Trans 1577–1588. Br Mycol Soc 90: 369–373. Wei H, Vienken K, Weber R, Bunting S, Requena N & Fischer Zonneveld BJM (1988b) Effect of carbon dioxide on fruiting in R (2004) A putative high afﬁnity hexose transporter, hxtA, Aspergillus nidulans. Trans Br Mycol Soc 91: 625–629.