16 Fruiting-Body Development in Ascomycetes

S. Pöggeler1,M.Nowrousian1,U.Kück1

CONTENTS I. Introduction I. Introduction ...... 325 A. Induction of Sexual Development andFruiting-BodyMorphology...... 326 The primary morphological character that dis- B. Model Organisms to Study Fruiting-Body tinguishes members of the phylum Ascomycota Development...... 327 1. Neurospora crassa ...... 327 from all other fungi is the ascus. This sac-like 2. Sordaria macrospora ...... 331 meiosporangium contains the meiospores (as- 3. Aspergillus nidulans ...... 331 cospores) and is produced only during the sexual II. Physiological Factors Influencing life cycle. The taxonomic classification of members Fruiting-Body Development ...... 333 A.EnvironmentalFactors...... 333 of the phylum Ascomycota is complex, because 1.NutrientsandRelatedFactors...... 333 these comprise a multitude of species showing 2.PhysicalFactors...... 334 high diversity in morphology, habitat and life B.EndogenousFactors...... 336 history. However, the ability to produce a dikaryon 1.MetabolicProcesses...... 336 separates members of the Ascomycota into two 2.Pheromones...... 338 III. Signal Transduction Cascades ...... 339 distinct groups: (1) the saccharomycetes, which are A. Perception of Environmental mostly unicellular, and (2) mycelial ascomycetes, andEndogenousSignals...... 339 the majority of which share several characteristics B.SignalTransductionPathways...... 341 including certain cell wall constituents, septal 1.HeterotrimericGProteins...... 342 2.RASandRAS-LikeProteins...... 342 pores with Woronin bodies, and a dikaryotic phase 3. cAMP Activated Protein Kinases PKA . 343 as part of their life cycle. Mycelial ascomycetes 4. Mitogen-Activated Protein Kinase typically form fruiting bodies called ascomata (MAPK)Pathways...... 343 or ascocarps (Alexopoulos et al. 1996; Barr 5. Other Putative Signaling Proteins . . . . 344 2001). These are highly complex, multicellular C.TranscriptionFactors...... 344 IV. Structural Components Involved structures composed of many different cell types in Fruiting-Body Development ...... 345 that surround the asci in a characteristic manner A. The Cytoskeleton (Bistis et al. 2003). Whereas ascospores arise inFruiting-BodyDevelopment...... 346 from dikaryotic hyphae after karyogamy and B. The Cell Wall inFruiting-BodyDevelopment...... 346 meiosis, all fruiting body-forming tissues rise 1.Chitin...... 346 from haploid, non-dikaryotic hyphae. This feature 2.Carbohydrates...... 346 clearly distinguishes fruiting-body formation in 3.Pigments ...... 347 ascomycetes from that in basidiomycetous fungi, 4.CellWallProteins...... 347 in which dikaryotic hyphae are involved in both 5. Are Multiple Genes with Overlapping Functions Involved the meiotic cycle and fruiting-body formation inCellWallMetabolism?...... 348 (for review, see Moore 1998). Fruiting-body V. C o n c lu s i o n s ...... 348 development in filamentous ascomycetes is a com- References...... 348 plex cellular differentiation process that requires special environmental conditions and is controlled by many developmentally regulated genes. This chapter gives a concise overview on the basics of fruiting-body development in hyphal ascomycetes, 1 Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Univer- focusing on aspects relevant for taxonomy and sität Bochum, 44780 Bochum, Germany morphology, and it summarizes environmental

The Mycota I Growth, Differentation and Sexuality Kües/Fischer (Eds.) © Springer-Verlag Berlin Heidelberg 2006 326 S. Pöggeler et al. as well as intrinsic signals influencing ascocarp ers), and divide mitotically in synchrony. The two formation. Finally, we highlight recent molec- nuclei remain in close association and undergo suc- ular genetic datasets from forward and reverse cessive divisions that result in dikaryotic cells (as- genetic approaches, which give further insight cogenous hyphae), in which each cell has two sexu- into presumptive signal transduction pathways ally compatible haploid nuclei. During the dikary- determining this multicellular differentiation otic phase, a dikaryotic mycelium grows within, process. and draws nourishment from, the haploid ascoma tissue. Inside the ultimate branches of the dikaryotic hyphae (the young asci), of which there may A. InductionofSexualDevelopment be millions in larger ascomata, fusion of the male and Fruiting-Body Morphology and female nucleus takes place. Immediately after karyogamy, the diploid nucleus of the zygote un- The sexual life cycle of ascomycetes can be ei- dergoes meiosis, which is in many species followed ther heterothallic (self-incompatible) or homothal- by a post-meiotic mitosis, resulting in the forma- lic (self-compatible). Heterothallic fungi exist in tionofeightnucleithatwillbecomeincorporated two mating types, designated, for example, A and into eight ascospores (see Zickler, Chap. 20, this a (or + and −), and mating occurs only between volume). sexual structures of opposite mating type. In a ho- Depending on the number of post-meiotic mi- mothallic fungus, every strain is able to complete toses and the degeneration of nuclei after meiosis, the sexual cycle without a mating partner. thenumberofascosporeswithinanascusvaries Sexual reproduction is typically controlled by between one and many thousands. The sizes and genes that reside in the mating-type locus. Mating shapes of ascospores and asci vary in a species- type-encoded proteins are putative transcription specific manner (Esser 1982; Alexopoulos et al. factors that are thought to act as transcriptional 1996). Based on light microscope studies, and ir- regulators on pheromone and pheromone recep- respective of size and shape, different types of asci tor genes (see Sect. III.C, and Debuchy and Tur- can be defined (Kirk et al. 2001). In a large number geon, Chap. 15, this volume). In most cases, the sin- of hyphal ascomycetes, the spores are discharged gle mating-type locus conferring mating behavior fromtheascus.Insidetheascoma(fruitingbody), consists of dissimilar DNA sequences (idiomorphs) the asci may be arranged in a scattered fashion or in the mating partners (Coppin et al. 1997; Kron- within a definite layer, which is called the hyme- stad and Staben 1997; Pöggeler 2001). nium. The first step in the sexual reproduction of Four major types of the multicellular asco- mycelial fungi is to combine two compatible nu- mata (fruiting bodies) can be distinguished: cleis- cleiinthesamecell.Thisoccursbyfusionofmor- tothecia, perithecia, apothecia and pseudothecia phologically similar or morphologically differenti- (Fig. 16.1). These produce the asci, and act as the ated gametangia, the male antheridia and female platformsfromwhichthemeiosporesarelaunched. ascogonia. In some species, the ascogonium is sur- The cleistothecium is a completely closed fruit- rounded by sterile hyphae to form a pre-fruiting ing body, whereas the perithecium is a more or body. It then bears a specialized hypha, the trichog- less closed, globose or flask-like fruiting body with yne, which receives the male nucleus. A functional a pore (ostiole) through which the ascospores es- male gamete may be a uninucleate spermatium cape. The apothecium is a cup- or saucer-like as- or microconidium or a multinucleate macroconid- coma in which the hymenium is exposed at ma- ium,whichcanbeformedonthemyceliumorin turity. In ascomycetes with a pseudothecium (or specialized structures called spermogonia. Subse- ascostroma), the fruiting body is initiated by the quenttocontactwithamalegamete,thetrichog- formation of a locule within a stroma, which is yne recruits a fertilizing nucleus from the male a matrix of vegetative hyphae, with or without tis- gamete, which migrates to the ascogonium (Bis- sue of a host. In contrast to cleistothecia, perithecia tis 1981). An alternative method of fertilization, and apothecia, the sexual organs of fungi produc- referred to as somatogamy, involves the fusion of ing pseudothecia are formed from hyphae already unspecialized somatic hyphae of two compatible within the developing ascocarp. In addition to asci, mycelia. Fertilization is not immediately followed many fruiting bodies contain sterile hyphae of var- by karyogamy. The nuclei migrate in pairs to the de- ious types that are important taxonomic charac- veloping, hook-shaped ascogenous hyphae (croiz- ters. Amongst these are paraphyses, elongated hy- Fruiting Bodies in Ascomycetes 327

Fig. 16.1. Fruiting bodies of ﬁla- mentous ascomycetes

phae originating from the base of an ascocarp, pe- pyrenomycetes Neurospora crassa and Sordaria riphyses, short unbranched hyphae in the ostiolar macrospora,andtheplectomyceteAspergillus canal of perithecia, and pseudoparaphyses, sterile nidulans, which will be described in detail in the hyphae originating above the level of asci in a pseu- following sections. However, molecular genetic dothecium (Alexopoulos et al. 1996). analyses of many other hyphal ascomycetes, Traditionally, fruiting-body morphology and including the pyrenomycetes Podospora anserina, the ascus structure have been used for a general Magnaporthe grisea and Cryphonectria parasitica taxonomic classification of the hyphal ascomycetes, as well as the loculoascomycete Cochliobolus which have thereby been grouped into four classes. heterostrophus,havealsoledtotheisolationof The typical ascocarps of plectomycetes, pyreno- fruiting-body developmental genes (Table 16.1). mycetes, discomycetes and loculoascomycetes are cleistothecia, perithecia, apothecia and pseudothe- 1. Neurospora crassa cia, respectively. However, this traditional classification has been questioned in recent years, due Since the 1940s, the pyrenomycete N. crassa has to the availability of molecular markers based on been used as model organism for genetic and DNA sequencing data (Berbee et al. 2000). From biochemical analyses (Perkins and Davis 2000; this follows that some groups, such as the pyreno- Davis and Perkins 2002). N. crassa is heterothal- mycetes and plectomycetes, can indeed be defined lic with two mating types, designated mat a by their ascocarp and ascus structure. However, and mat A, and is able to propagate by both others like the discomycetes and loculoascomycetes sexual and asexual spores. Haploid strains are appear not to be monophyletic (Liu et al. 1999; hermaphroditic but self-sterile. The formation of Lumbsch 2000; Lumbsch et al. 2000; Lindenmuth protoperithecia is induced by nitrogen limitation. et al. 2001). However, the sexual cycle is initiated only when a protoperithecium of one mating type is fertilized via a trichogyne by a male cell of another mating B. Model Organisms to Study Fruiting-Body type. In N. crassa, there are no specialized male Development cells that function exclusively as spermatia; rather, macroconidia, microconidia or hyphal fragments In recent years, classical and molecular genetic ap- can fertilize the protoperithecia (see Glass and proaches have been used to gain a detailed insight Fleißner, Chap. 7, this volume). Fertilized pro- into the mechanism directing the cellular processes toperithecia develop into perithecia within which of fruiting-body development. asci with eight linearly ordered, homokaryotic Classical genetic studies have demonstrated ascospores are formed. Over 200 mutants that that fruiting-body development is under polygenic affect sexual development have been isolated in N. control. Much information has come from muta- crassa. These include male-sterile mutants that are genesis assessments in which mutants have been fertile as female parents, female-sterile mutants obtainedthatareblockedatonespecificstageof that are fertile as male parents, mutants that development. Fungal model organisms used to are both female- and male-sterile, and mutants study fruiting-body development are mainly the that affect ascus development in various ways 328 S. Pöggeler et al.

Table. 16.1. Genes involved in fruiting-body development

Organisms Gene Gene product Defect of mutants Reference Neurospora asd-1 Rhamnogalacturonase No ascospores Nelson et al. (1997b) crassa ndk-1 Nucleoside diphosphate Light-dependent Ogura et al. (2001) kinase positioning of perithecia sod-1 Superoxid dismutase Light-dependent Yo s h i d a a n d positioning of perithecia Hasunuma (2004) nuo12.3, Subunit of respiratory No ascospores Fecke et al. (1994), nuo20.8, chain NADH dehydrogenase Duarte et al. (1998), nuo30.4, Almeida et al. (1999), nuo78, Duarte and Videira (2000) nuo51, nuo24, nuo2 mfa-1 Hydrophobic peptide Male sterility, aberrant Kim et al. (2002a) pheromone female sexual development and ascospore production pre-1 GPRC, pheromone receptor Female sterility Kim and Borkovich (2004) gna-1 Gproteinα subunit Female-sterile, no perithecia Ivey et al. (1996), Yang and Borkovich (1999) gna-3 Gproteinα subunit Smaller perithecia with no beaks, Kays et al. (2000) reduced number of ascospores gnb-1 Gproteinβ subunit Female-sterile, no perithecia Yang et al. (2002) gng-1 Gproteinγ subunit Female-sterile, no perithecia Krystofova and Borkovich (2005) krev-1 GTP/GDP-binding protein; No perithecia Ito et al. (1997) member of the ras superfamily cr-1 Adenylyl cyclase Delayed perithecia Perkins et al. (1982), and ascospore formation Ivey et al. (2002) mak-2 MAPK, related Female-sterile, Pandey et al. (2004), to S. cerevisiae Fus3p no protoperithecia Li et al. (2005) and Kss1p nrc-1 MAPKKK, similar Female-sterile, Kothe and Free (1998) to S. cerevisiae Ste11p no protoperithecia nrc-2 Serine-threonine protein Female-sterile, Kothe and Free (1998) kinase no protoperithecia rgb-1 B regulatory subunit of the Female-sterile, Yatzkan and Yarden (1999) type 2A Ser/Thr phosphatase no prothoperithecia ham-2 Putative transmembrane Female-sterile, Xiang et al. (2002) protein no protoperithecia eat-2 Highlysimilartoadomain Female-sterile, Randall and present in the plasma no prothoperithecia Metzenberg (1998) membrane ATPase cel-2 β subunit of fatty Fewer peritecia, rare ascospores Goodrich-Tanrikulu acid synthase et al. (1999) asm-1 Transcription factor with Female-sterile, Aramayo et al. (1996) APSES domain no protoperithecia vma-1 Subunit A of the V-ATPase Female-sterile, Bowman et al. (2000) no protoperithecia, can not donate nuclei in a cross cpc-2 WD-repeat protein required Female-sterile, Müller et al. (1995) to repress general amino no protoperithecia acid control, scaffold protein rco-1 Multidomain protein that Female-sterile, Yamashiro et al. (1996) mediates transcriptional no protoperithecia repression asd-4 GATA-type zinc ﬁnger No asci, no ascospores Feng et al. (2000) transcription factor wc-1 GATA-like zinc ﬁnger No phototropism Ballario et al. (1996), transcription factor of perithecial beaks Oda and Hasunuma (1997) Fruiting Bodies in Ascomycetes 329

Table. 16.1. (continued)

Organisms Gene Gene product Defect of mutants Reference wc-2 GATA-like zinc finger No phototropism Linden and Macino (1997), transcription factor of perithecial beaks Oda and Hasunuma (1997) pp-1 Transcription factor No protoperithecia Li et al. (2005) homeodomain, C2H2 zinc finger, Ste12p homolog Podospora car1 Peroxisomal membrane Impaired karyogamy, Berteaux-Lecellier et al. anserina protein no ascospores (1995) cro1 Cytosolic protein Impaired meiosis, Berteaux-Lecellier et al. with C2H2 zinc finger motif no ascospores (1998) AS4 Translation-elongation No ascospores, Silar et al. (2001) factor EF-1α, eF1A no perithecia su1 Translation-termination Female-sterile, Gagny and Silar (1998) factor eRF3 no protoperithecia su2 Translation-termination Female-sterile, Gagny and Silar (1998) factor eRF1 no protoperithecia PaCox17 Chaperone targeting copper Delayed perithecial formation Stumpferl et al. (2004) to cytochrome c and reduced ascus formation rmp1 No homology, functions Lethal or without mitochondrial Contamine et al. (2004) in nucleus–mitochondria targeting sequence, no ascospores cross-talk PaNox1 NADPH oxidase No perithecia Malagnac et al. (2004) PaNox2 NADPH oxidase No ascospore germination Malagnac et al. (2004) mod-A Proline-rich protein Female-sterile, Barreau et al. (1998) with SH3-binding motif no protoperithecia mod-E Heat-shock protein HSP90 Sterile perithecia, no ascospores Loubradou et al. (1997) mfp Hydrophobic peptide Male-sterile Coppin et al. (2005) pheromone mfm Peptide pheromone Male-sterile Coppin et al. (2005) mod-D Gproteinα subunit Female-sterile, Loubradou et al. (1999) no protoperithecia PaAsk1 MAPKKK, similar No perithecia Kicka and Silar (2004) to S. cerevisiae Bck1p grisea Copper-activated Female-sterile, Osiewacz and transcription factor, no protoperithecia Nuber (1996), ortholog of the yeast Borghouts and transcription factor MAC1 Osiewacz (1998) fle1 Transcription factor, Fewer microconidia, more Coppin (2002) C2H2 zinc finger abundant protoperithecia pah1 Transcription factor, Enhanced number of Arnaise et al. (2001) homeobox protein microconidia, delayed protoperithecia development ami1 Homolog of A. nidulans Male-sterile, delayed Graia et al. (2000) APS-A fruiting-body formation Sordaria pro11 WD-repeat protein No perithecia Pöggeler and Kück (2004) macrospora pro4 Leucine biosynthesis No perithecia Kück (2005) acl1 Subunit of ATP-citrate-lyase No ascospores Nowrousian et al. (1999) spo76 Chromosomal protein Impaired meiosis, few ascospores van Heemst et al. (1999) pro1 C6 zinc finger transcription No perithecia Masloff et al. (1999) factor Aspergillus trpB Tryptophane biosynthesis No cleistothecia Eckert et al. (1999, 2000) nidulans hisB Histidine biosynthesis No cleistothecia Busch et al. (2001) tubB α-tubulin No ascospores Kirk and Morris (1991) uvsC DNA-repair enzyme No karyogamy, no ascospores van Heemst et al. (1997) homolog of S. cerevisiae RAD51 lsdA No sequence similarity, No inhibition of cleistothecia Lee et al. (2001) unknown function development under high salt conditions 330 S. Pöggeler et al.

Table. 16.1. (continued)

Organisms Gene Gene product Defect of mutants Reference phoA Cyclin-dependent kinase Enhanced number of cleistothecia Bussink and Osmani (1998) under phosphorus-limited conditions pho80 Putative cyclin-dependent Promotes sexual development Wu et al. (2004) kinase inhibitor csnD Subunit of COP9 signalosome No cleistothecia Busch et al. (2003) veA No homology, unknown No cleistothecia Kim et al. (2002b) noxA NADPH oxidase No cleistothecia Lara-Ortíz et al. (2003) ppoA Putative fatty acid Increased ratio of asexual Tsitsigiannis et al. (2004) dioxygenase to sexual development odeA ∆-12 desaturase Delayed ascosporogenesis Calvo et al. (2001) gprA GPRC, pheromone receptor Fewer cleistothecia and ascospores Seo et al. (2004) gprB GPRC, pheromone receptor Fewer cleistothecia and ascospores Seo et al. (2004) gprD GPRC No cleistothecia Han et al. (2004) fadA Gproteinα subunit No cleistothecia Rosèn et al. (1999) sfdA Gproteinβ subunit No cleistothecia, more Hülle cells Rosèn et al. (1999) ﬂbA RGS protein No cleistothecia Han et al. (2001) sakA MAPK, similar Premature sexual development, Kawasaki et al. (2002) to S. cerevisiae Hog1p more cleistothecia steC MAPKKK, similar No cleistothecia Wei et al. (2003) to S. cerevisiae Ste11p steA Transcription factor No cleistothecia, Vallim et al. (2000) homeodomain, C2H2 zinc only Hülle cells ﬁnger, Ste12p homolog nsdD Transcription factor No cleistothecia, no Hülle cells Han et al. (2001) GATA-type stuA Transcription factor No cleistothecia, no Hülle cells Wu and Miller (1997) with APSES domain dopA Putative transcription No cleistothecia, no Hülle cells Pascon Castiglioni and factor, leucine zipper-like Miller (2000) domains and similarity to C/EBP transcription factor medA Transcriptional regulator No cleistothecia, only Hülle cells Busby et al. (1996) cpcA Transcription factor, When overexpressed, Hoffmann et al. (2001b) c-Jun-like leucine zipper, no cleistothecia required to activate general amino acid control cpcB WD-repeat protein, required No cleistothecia Hoffmann et al. (2000) to repress general amino acid control, scaffold protein Magnaporthe magA Gproteinα subunit No ascospores Liu and Dean (1997) grisea magC Gproteinα subunit No ascospores Liu and Dean (1997) magB Gproteinα subunit Female-sterile, no perithecia Liu and Dean (1997) mac-1 Adenylyl cyclase Female-sterile, no perithecia Choi and Dean (1997) mps-1 MAPK, similar Female-sterile, no perithecia Xu et al. (1998) to S. cerevisiae Sltp Cryphonectria Mf2-2 Hydrophobic peptide Female sterility Zhang et al. (1993) parasitica pheromone Mf1-1 Peptide pheromone Male-sterile Turina et al. (2003) cpg-1 Gproteinα subunit Female-sterile Gao and Nuss (1996) cpg-2 Gproteinα subunit Enhanced perithecial development Gao and Nuss (1996) Fusarium mgv-1 MAPK, similar Female-sterile, no perithecia Hou et al. (2002) graminearum to S. cerevisiae Sltp Cochliobolus cga-1 Gproteinα subunit Female sterility Horwitz et al. (1999) heterostrophus chk-1 MAPK, related to Female sterility Lev et al. (1998) S. cerevisiae Fus3p and Kss1p Fruiting Bodies in Ascomycetes 331

(Raju 1992). When male-sterile mutants are used 2. Sordaria macrospora as fertilizing parent, perithecial development is initiated, but then arrested at an early stage. In the heterothallic species N. crassa,mutations In N. crassa, numerous female-sterile mutants conferring male and/or female sterility can be that do not form functional protoperithecia or detected directly because of their sterility effects display a reduced fertility have been described. in heterozygous crosses. However, recessive mu- Because of this high frequency of sterility in tations that affect post-fertilization perithecial female strains, it was suggested that at least 400 development will remain undetected in heterothal- genes are required for perithecium and ascospore lic species until the mutant allele is available in development in N. crassa (Johnson 1978; Leslie and both mating types, thus allowing homozygous Raju 1985). However, many of these female-sterile crosses. In contrast to N. crassa, the homothallic mutants show abnormal vegetative growth, and pyrenomycete S. macrospora is self-fertile, which thus this female sterility may be a consequence means that recessive mutations can directly be of a morphological defect, rather than a mutation tested for defects in fruiting-body development. in a gene specifically needed for fruiting-body Moreover, S. macrospora produces only meioti- differentiation (Raju 1992). cally derived ascospores, whereas asexual spores, Effective molecular techniques developed for such as conidia, are absent. Thus, there is no N. crassa, such as the transformation and creation interference between two different developmental of cosmid genomic libraries involving, for exam- programs,whichmakesiteasier,forexample,to ple, phenotypic complementation, have led to the analyze differentially expressed genes involved in cloning and functional characterization of regu- ascocarp development. Under laboratory condi- latorygenesthataffectmorphology(Baileyand tions, perithecia and ascospores reach maturity Ebbole 1998). Other molecular genetic approaches within 7 days after ascospore germination. During used subtractive hybridization to isolate N. crassa this development, distinct reproductive structures, sexual developmental genes. such as ascogonia, protoperithecia (young fruiting With this attempt, Nelson and Metzenberg bodies), and perithecia, can be distinguished (1992) succeeded in identifying 14 genes tran- (Fig. 16.2). Because S. macrospora represents scribed only under nitrogen-depleting growth such a favorable genetic system for scientists, this conditions. One of these, asd-1,hasbeenshownto homothallic pyrenomycete was used to generate encode a putative rhamnogalacturonase necessary numerous mutants that are blocked at various for ascus development (Nelson et al. 1997b). stages of perithecial development (Esser and In large-scale analyses, fruiting body-specific Straub 1958; Masloff et al. 1999). Wild-type strains expressedsequencetags(ESTs)fromN. crassa of S. macrospora areself-fertileandproduce were sequenced for further molecular characteri- perithecia. However, fertile perithecia are also zation (Nelson et al. 1997a). In such experimental formed in crosses between sterile strains, when approaches, isolated candidate genes have to be nuclei are interchanged by hyphal anastomoses inactivated to obtain detailed information about at the contact zones of two sterile mycelia. These mutant phenotypes and gene function. crosses facilitate the analysis of epistatic rela- Inactivation of developmental genes in N. tionships among developmental mutants. The crassa was achieved either by homologous recom- establishment of molecular tools provides the bination or by gene silencing via repeat-induced basis for studying fruiting-body development in S. point mutation (RIP). The N. crassa RIP process macrospora (Walz and Kück 1995; Pöggeler et al. efficiently detects and mutates both copies of a se- 1997). quence duplication. RIP acts during the dikaryotic stage of the sexual cycle, causing numerous C:G- 3. Aspergillus nidulans to-T:A transitions within duplicated sequences, andisfrequentlyusedtoinactivategenesinN. The plectomycete Aspergillus nidulans (teleo- crassa (Galagan and Selker 2004). Finally, the morph: Emericella nidulans)propagatesbythe whole genome sequence of N. crassa has become formation of spores that can be either asexual or available, opening the opportunity for analyzing sexual,andhaslongservedasamodelsystemfor genes involved in fruiting-body development by understanding the genetic regulation of asexual means of reverse genetic approaches (Galagan development in ascomycetes (Adams et al. 1998). et al. 2003; Borkovich et al. 2004). Theasexualcycleischaracterizedbytheproduc- 332 S. Pöggeler et al.

In contrast to pyrenomycetes, the presumptive ascogonium of A. nidulans is not surrounded by sterile hyphae, and pre-fruiting bodies are not formed. Only after fertilization is the ascogonium surrounded by growing, unordered hyphae, which form an increasingly packed “nest” and then differentiate globose, multinucleate Hülle cells, which support the development of cleistothecia (Ellis et al. 1973). The surrounding hyphae that form the “nest” later differentiate into the cleistothecial envelope. The developing spherical cleistothecia are filled with ascogenous hyphae that differentiate into asci. After karyogamy, the zygote stage is immediately followed by meiosis. Directly after meiosis, the four resulting nuclei pass through a first post-meiotic mitosis. The eight nuclei are then separated by membranes, and give rise to eight red-pigmented ascospores within each ascus. As second post-meiotic mitosis results in eight binucleate, mature ascospores (Braus et al. 2002). Similarly to the homothallic pyrenomycete S. macrospora,self-fertileA. nidulans strains can be used in crossing experiments. In this case, hyphae from two different strains can form anastomoses and exchange nuclei when growing sufficiently close to each other (Hoffmann et al. 2001a). Fig. 16.2. Sexual development of Sordaria macrospora. A,B The coexistence of sexual and asexual repro- Youngascogonium;C,Dascogonium;E,Fprotoperithecium; duction within one and the same individual has G perithecium; H top view of perithecium, with ascospores. made A. nidulans a popular genetic model organ- A,C,E Differential interference contrast light micrographs ism to compare fitness effects of sexual and asexual DIC. B,D,F Fluorescence micrographs of A,C,andE with DAPIstainingofnuclei.G,HScanningelectron micrographs reproduction. Because of its homothallism, the sexual and asexual offspring of A. nidulans have largely identical genotypes. As was shown by Bruggeman et al. (2003, 2004), slightly deleterious mutations tion of haploid conidiophores that bear asexual, accumulate at a lower rate in the sexual than in the single-celled spores called conidia (Fischer 2002, asexual pathway. see Fischer and Kües, Chap. 14, this volume). In addition to classical genetics, various Sexual development of A. nidulans starts after tools required for molecular biology have been conidiophore differentiation. The conidiophores developed, and recently the entire genome of A. are produced by mitotic division in 3 days after nidulans has been sequenced (http://www.broad. germination, whereas the sexual ascospores are mit.edu/annotation/fungi/aspergillus), thus mak- formed after at least 7 days (Pontecorvo 1953). ing A. nidulans an excellent model for studying A. nidulans is homothallic, and a single colony various biological questions, including the can produce cleistothecia filled with up to 1000 multicellular cleistothecium development. ascospores by self-fertilization. For the genetic dissection of sexual sporula- In A. nidulans, no obvious antheridium or as- tion in A. nidulans, a collection of ascospore-less cogonium structures can be observed (Benjamin mutants was isolated by Swart et al. (2001). To un- 1955). It was, however, assumed by Kwon and Raper derstand the sexual reproduction of A. nidulans in (1967) that in Aspergillus heterothallicus, a het- more detail, Han et al. (1990) identified numerous erothallic relative of A. nidulans,acoiledstructure mutants that were defective in sexual development equivalent to an ascogonium fuses to a second cell in a forward genetic screen. These were classified equivalent to an antheridium. into two groups: Fruiting Bodies in Ascomycetes 333

1. nsd (never in sexual development) mutants that 1992). In this section, a brief overview of some of are unable to form any sexual structures; and these factors is given, focusing on cases where at 2. bsd (block in sexual development) mutants that least some genes involved in the perception of these show differences in the amount or timing of factors have been identified. sexual organ production, compared to the wild type. 1. Nutrients and Related Factors Several nsd mutants were analyzed for their ge- The requirements for certain nutrients and other netic and morphological characteristics (Han et al. chemical substances for fruiting-body formation 1994, 1998). Finally, the nsdD gene has been cloned vary in different ascomycetes. In many species, and was shown to encode a GATA-type transcrip- fruiting bodies are formed preferentially at tion factor (Han et al. 2001). Beside the nsdD gene, much lower nutrient concentrations than those several other A. nidulans genesthataffectsexual promoting vegetative growth (Moore-Landecker development have been cloned, not only by com- 1992). One reason might be that the vegetative plementation of developmental mutants but also by mycelium accumulates nutrients that can be used reverse genetic approaches (Table 16.1). for the formation of fruiting bodies once a critical amount is reached. Thus, the mycelium would, in effect, nurture the developing fruiting bodies. II. Physiological Factors Influencing Another reason could be that sexual development Fruiting-Body Development is initiated when the growth substrate is depleted of nutrients, and durable ascospores are produced Most fungi do not form fruiting bodies contin- that can lie dormant until more suitable conditions uously, but require special environmental condi- occur. tions. Additionally, the vegetative mycelium has Some ascomycetes require specific factors, to acquire a certain stage of “competence” before such as vitamins, for sexual development, but differentiating fruiting bodies. The external fac- not for vegetative growth. S. macrospora,for tors needed to promote fruiting-body formation example, needs biotin to complete the sexual are mostly organism-specific, and depend on the cycle (Molowitz et al. 1976). Furthermore, it ecological niche the fungus occupies. Most promi- has been shown in S. macrospora that addition nent among these are nutrients, light, tempera- of arginine to the growth medium enhances ture, aeration, pH, and the presence of a partner fruiting-body formation (Molowitz et al. 1976). or host for symbiotic or pathogenic fungi, respec- This finding, together with the observation that tively. Endogenous factors that have been identi- amino acid synthesis mutants of both A. nidulans fied as relevant for fruiting-body formation often and S. macrospora show defects in fruiting-body involve components of primary metabolism as well development, indicates an influence of amino as pheromones or hormone-like substances (Dyer acid metabolism in sexual development, an aspect et al. 1992; Moore-Landecker 1992). Perception of discussed in more detail in Sect. II.B.1. In some these factors and initiation of an appropriate re- ascomycetes, fruiting-body formation can be action require complex regulatory networks; in re- increased by addition of fatty acids to the growth cent years, genetic and molecular biology methods medium. In Ceratocystis ulmi and Nectria haema- have helped to start unraveling the molecular basis tococca, production of perithecia can be increased of fruiting-body development. by exogenous linoleic acid, whereas in N. crassa, both oleate and linoleate enhance fruiting-body formation (Nukina et al. 1981; Marshall et al. 1982; A. Environmental Factors Dyer et al. 1993; Goodrich-Tanrikulu et al. 1998). Mutants with defects in lipid metabolism often The influence of environmental factors on fruiting- have developmental defects, too (see Sect. II.B.1). body development is well-studied on the physio- Environmental factors that influence sexual logical level in economically important fungi, most development(andeachother)arethepHofthe of which are basidiomycetes (Kües and Liu 2000), growth medium, aeration (CO2 pressure), osmotic and to some degree in several ascomycetes, which pressure, and the presence and availability of min- have been used to investigate the basic principles eral salts. In N. crassa, mutants in a gene encoding of fruiting-body development (Moore-Landecker a subunit of the vacuolar H(+)-ATPase do not grow 334 S. Pöggeler et al. in medium with a pH of 7.0 and above, and they molecular mechanisms of development (Nowrou- are female-sterile, most likely due to insufficient sian et al. 2004). Lacourt and coworkers compared cellular homeostasis (Bowman et al. 2000). It has the expression of 171 genes in vegetative tissue long been known that in A. nidulans,increased and different stages of developing fruiting bodies partial pressure of carbon dioxide favors fruiting- of the mycorrhizal ascomycete Tuber borchii, body formation, whereas aeration, and thereby using cDNA macroarrays. These investigations the removal of carbon dioxide, shifts the balance revealed that metabolism and cell wall synthesis toward asexual sporulation (Champe et al. 1994). are substantially altered during development In A. nidulans, high concentrations of potas- (Lacourt et al. 2002), but how the plant partner sium, sodium or magnesium ions inhibit sexual influences fungal development at the molecular development and promote asexual sporulation. level remains to be elucidated. Regulation of this salt-dependent developmental balance requires the lsdA gene, which does not 2. Physical Factors haveanyhomologytopreviouslycharacterized genes (Lee et al. 2001). Also involved in this Most ascomycetes form fruiting bodies on the sur- regulation process is veA, a gene that integrates face of their growth substrate to facilitate ascospore anumberofsignalsandthatisdescribedmore dispersal, the most notable exception being truf- fully in Sect. II.A.2. fles and related fungi. This means that the fun- Another gene involved in salt-dependent de- gussomehowhastoorganizethecorrectplace velopmental decisions between asexual and sex- and orientation of the fruiting bodies. Possible ual development in A. nidulans is phoA,agene mechanisms for this process include the percep- for a cyclin-dependent kinase. Depending on both tion of gravity or of air/substrate interfaces. There pH and the initial phosphorus concentration, phoA has been some research into the effects of grav- mutants can switch from asexual to sexual devel- ity on fungal sexual development that has estab- opment, or do not show any form of spore differ- lished that at least some fungal fruiting bodies, or entiation (Bussink and Osmani 1998). Interacting parts of fruiting bodies exhibit gravitropism, but with PHOA is the cyclin An-PHO80, which also is the genetic and biochemical processes needed to involved in regulating the balance between asexual perceive and respond to gravity remain enigmatic and sexual differentiation. Effects of the An-pho80 (see Corrochano and Galland, Chap. 13, this vol- deletion also depend on phosphate concentration, ume). Little more is known about the recognition butincontrasttophoA mutants, An-pho80 mutants of air/surface interfaces, or other means of spatial increase the production of conidia and do not form control of fruiting-body formation in ascomycetes. mature cleistothecia (Wu et al. 2004). In basidiomycetes, a class of small secreted proteins Many pathogenic or mycorrhizal fungi form called hydrophobins has been shown to be essen- fruiting bodies only in contact with their hosts tial for breaching an air/water interface, and hy- or symbiont partners, which makes analysis drophobins coat many fruiting-body surfaces (see of these differentiation processes difficult. For Chap. 19, this volume). Hydrophobins have been a taxol-producing Pestalotiopsis microspora isolate identified in several ascomycetes, too, but whether thatlivesasanendophyteofyew,itwasfound they play a role in fruiting-body formation is not yet that a methylene chloride extract of yew needles clear (see Sect. IV.B). Another effect that has been induces the formation of perithecia (Metz et al. observed in several ascomycetes is the so-called 2000). Most likely, one or more lipid-like com- edge effect – fruiting bodies are formed preferen- pounds present in yew needles stimulate sexual tially at the edges of a petri dish or other culture differentiation in the fungus. vessels. Investigation of this effect in S. macrospora Lipid-derived factors produced by fungi them- has revealed that the determining parameter is not selves, as well as lipid metabolism in general have a recognition of edges or other surface structures, alsobeenfoundtobeinvolvedinsexualdevelop- but rather an increased hyphal density, which can ment (see Sect. II.B.1). beduetomechanicalobstaclesandalsotonutri- For organisms which cannot easily be cul- ent availability (Molowitz et al. 1976; Hock et al. tivated under laboratory conditions, but where 1978); similar results have been reached with other RNA extraction is possible from field isolates, ascomycetes (Moore 1998). large-scale expression analysis techniques such as Relatedtothe“edgeeffect”mightbetheob- microarrays have great potential to help unraveling servation that fruiting bodies are often formed at Fruiting Bodies in Ascomycetes 335 regular intervals; both effects require the fungus candidate for a light signal transduction pathway to sense hyphal or fruiting-body spacing, but the component (Ogura et al. 2001). genetic basis behind this regulation remains to be Studies with double mutants have shown that elucidated. ndk-1 depends on the presence of functional wc Another possible signal for the production genes (Yoshida and Hasunuma 2004). Both WC and orientation of fruiting bodies is light (see proteins are also part of the circadian clock of N. also Chap. 13, this volume). Light has been crassa (Loros and Dunlap 2001), and transcription shown to influence fruiting-body formation in ofthepheromoneprecursorgenesthatareinvolved many ascomycetes from different phylogenetic in sexual development of N. crassa (see Sect. B.2) groups. Influences of light range from complete is regulated by the endogenous clock (Loros et al. light-dependence of fruiting-body formation 1989; Bobrowicz et al. 2002). These findings indi- to orientation of fruiting bodies or, in reverse, cate that in N. crassa, sexual development is not a preferential formation of fruiting bodies in the only light-regulated but the circadian clock may dark. Several genes involved in light perception also contribute to the control of fruiting-body for- have been identified in various fungi. mation (see also The Mycota, Vol. III, Chap. 11). Early reports of light effects on fruiting-body Theinfluenceoflightondevelopmentalpro- formation have been for Pyronema confluens cesses was also investigated extensively in A. nidu- and Pyronema domesticum, where apothecium lans. In this ascomycete, red light shifts the ratio of formation is completely light-dependent, and asexual to sexual reproduction structures (conidio- dark-grown mycelia are sterile (Claussen 1912; phores vs. cleistothecia) toward asexual reproduc- Moore-Landecker 1979). In other fungi, like N. tion, whereas fruiting-body formation is favored in crassa, fruiting bodies are formed in the dark and thedark. Onegenethatisnecessaryformaintaining are placed on the surface of the growth substrate this light-dependent balance is veA (Mooney and even without illumination, but perithecial necks Yager 1990; Kim et al. 2002b). veA,whichdoesnot are normally oriented toward the light, and in have homology to any genes with known function, darkness point to various directions (Harding is essential for fruiting-body formation; veA dele- and Melles 1983). Furthermore, not only the tion mutants do not form any cleistothecia (Kim orientation but also the position of the neck on et al. 2002b). The veA1 allele, a partially deleted the perithecium is light-dependent (Oda and form of veA that is present in many laboratory Hasunuma 1997). Additionally, the number of pro- strains of A. nidulans, causes a less severe pheno- toperithecia that are formed is greatly increased type with a preference for conidiation, even without upon blue-light illumination (Degli Innocenti illumination (Kim et al. 2002b). and Russo 1983). All three effects were shown to Another gene involved in controlling the light- depend on the white collar genes wc-1 and wc-2 dependent balance of asexual versus sexual repro- (Harding and Melles 1983; Degli Innocenti and duction of Aspergillus is csnD.Thisgeneencodes Russo 1984; Oda and Hasunuma 1997). WC-1 has asubunitoftheCOP9signalosome,aconserved been identified as a blue-light photoreceptor as eukaryotic protein complex that regulates develop- well as a transcription factor, and it interacts with mental processes by targeting proteins for ubiqui- WC-2 to regulate the transcription of target genes tinylation and subsequent degradation by the 26S in a light-dependent manner (Ballario et al. 1996; proteasome (Busch et al. 2003). In contrast to veA Linden and Macino 1997; Froehlich et al. 2002; He mutants, a csnD deletion mutant predominantly in- et al. 2002). duces(butdoesnotcomplete)thesexualcycle,irre- Another gene that plays a role in certain aspects spective of the light signal (Busch et al. 2003). How- of light-dependent morphogenesis is ndk-1 (Ogura ever, both veA and csnD mutants have other, light- et al. 2001; Yoshida and Hasunuma 2004). ndk-1 independent phenotypes, and may be part of signal encodes a nucleoside diphosphate kinase, and is transduction networks that integrate many signals required for light-dependent neck positioning on controlling fruiting-body development (Kim et al. theperitheciabutnot for orientation of theneckit- 2002b; Busch et al. 2003). self. Nucleoside diphosphate kinase is a conserved Another physical factor influencing fruiting- enzyme that is involved in signal transduction cas- body development is temperature. For those asco- cades in various organisms. mycetes in which this aspect has been investigated, In N. crassa, NDK-1 is autophosphorylated af- it was found that the temperature range that ter blue-light illumination, which makes it a likely controls fruiting-body formation is usually more 336 S. Pöggeler et al. restricted than that controlling vegetative growth also comprises a control point for progression of (Moore-Landecker 1992), but the genetic basis for sexual development (Hoffmann et al. 2000). This this is not yet clear. mod-E, a heat-shock protein was demonstrated by investigating the functions HSP90homolog,wasfoundtobeinvolvedinboth in fruiting-body formation of two members of sexual development and vegetative incompatibility the cross-pathway network, cpcA and cpcB. cpcA in Podospora anserina (Loubradou et al. 1997). encodes a transcriptional activator homologous to mod-E transcripts are accumulated after a shift the yeast Gcn4p protein, which is the activating from 26 to 37 ◦C, but effects of different temper- transcription factor for cross-pathway control atures on fruiting-body formation in the wild (termed general control of amino acid biosyn- type versus mod-E mutants were not reported. thesis) in Saccharomyces cerevisiae (Hoffmann Therefore, it remains to be determined whether et al. 2001b). CpcB is homologous to mammalian mod-E or other (heat-shock) proteins are involved RACK1 (receptor for activated C-kinase 1), a scaf- in temperature-dependence of fruiting-body foldproteininvolvedinmanycellularsignaling development. processes (McCahill et al. 2002). cpcA and cpcB play antagonistic roles in cross-pathway control as well as in sexual development: cpcA activates B. Endogenous Factors amino acid biosynthesis gene transcription under conditions of amino acid deprivation, whereas Thetransitionfromvegetativegrowthtosexualde- cpcB represses the cross-pathway control network velopment requires a physiologically “competent” when amino acids are present. Overexpression mycelium. This competence often depends on nu- of cpcA in the presence of amino acids leads to trient availability, but the nutrients also have to be a block in sexual development, thereby mimicking processed by the fungal metabolism; and genetic a lack of amino acids, and the same effect can be analyses have shown that fruiting-body formation reached by deletion of cpcB (Hoffmann et al. 2000). requires metabolic reactions different from those The connection between cross-pathway control of vegetative growth (see Sect. II.B.1). In several and sexual development seems to be widespread fungal species, pheromones or hormone-like sub- in filamentous ascomycetes, as a mutant in the stances are necessary for completion of the sexual N. crassa cpcB homolog, cpc-2, is female-sterile cycle, as described in Sect. II.B.2 and in Chap. 11 (Müller et al. 1995). Also, a sterile mutant of (this volume). S. macrospora was shown to have a defect in a gene for leucine biosynthesis (Kück 2005). This 1. Metabolic Processes mutant, as well as the A. nidulans amino acid biosynthesis mutants mentioned above, grow In several ascomycetes, it was found that mutations normally on media with moderate amounts of in genes for primary metabolism often interfere the amino acid they are auxotrophic for, but if at with sexual development under conditions where all, fertility can be restored only by much higher vegetative growth remains more or less normal. amounts. These findings indicate that fungi are Examples for this are mutants blocked in amino able to integrate nutrient availability and cellular acid biosynthesis pathways, and fatty acid biosyn- metabolism, and react properly with respect to the thesis mutants. Many effects of mutations leading initiation of energy-demanding processes such as to amino acid auxotrophy on fruiting-body mor- fruiting-body formation. phogenesis have been investigated in A. nidulans. Similar regulatory events can be proposed for Deletion of the tryptophan synthase-encoding fatty acid metabolism and fruiting-body develop- gene trpB, or the histidine biosynthesis gene ment, although the evidence here is more spuri- hisB leads to loss of cleistothecia production ous and signal transduction pathways have yet to on medium with low levels of tryptophan or be identified. Nevertheless, data from mutants in histidine, respectively (Eckert et al. 1999, 2000; diverse genes involved in different aspects of fatty Busch et al. 2001). Both genes are regulated by acid metabolism indicate that appropriate amounts the cross-pathway control system, a regulatory and composition of fatty acids and their deriva- network that activates a variety of amino acid tives are essential for sexual development. N. crassa biosynthesis genes when the amounts of a single mutants of a fatty acid synthase subunit are ster- amino acid are low. Besides regulating amino ile in homozygous crosses, and A. nidulans mu- acid biosynthesis, this cross-pathway network tants of several desaturase genes show changes in Fruiting Bodies in Ascomycetes 337 the balance between sexual and asexual develop- the developmental phenotype (Duarte and Videira ment (Goodrich-Tanrikulu et al. 1999; Calvo et al. 2000). This is consistent with an increased demand 2001; Wilson et al. 2004). In A. nidulans, several for energy and metabolites during fruiting-body fatty acid-derived factors, so-called psi factors, are formation. necessary for correct developmental decisions, and Another aspect of increased respiration is the changes in fatty acid composition also influence psi generation of reactive oxygen species (ROS), and factor composition, which could explain morpho- thereby oxidative stress. It has long been known logical defects in the mutants (see Chap. 11, this that ROS can have a deleterious effect on many cel- volume). It remains to be determined if similar lular compounds, e.g., DNA and proteins, but only fatty acid-derived specific developmental factors lately has it become acknowledged that the gener- are present in other ascomycetes. Analyses of other ation of ROS can be an actively regulated process, mutants point to a more general requirement for and that ROS can have diverse roles in cell physiol- fatty acids, probably as energy source for fruiting- ogy and signaling. body formation. In S. macrospora,thesterilemu- In N. crassa,itwasshownrecentlythatsod-1, tant per5 was found to harbor a defect in the acl1 which encodes a superoxide dismutase, is neces- gene that encodes a subunit of ATP citrate lyase sary for light-dependent positioning of perithecial (Nowrousian et al. 1999). This enzyme is involved necks (Yoshida and Hasunuma 2004). Superoxide in the production of cytosolic acetyl-CoA, which dismutases catalyze the conversion of oxygen radi- is used mainly for the synthesis of fatty acids and cals that are generated during aerobic metabolism sterols, and the mutant can be partially rescued to hydrogen peroxide, thereby protecting the or- by addition of exogenous oleate, thereby indicat- ganism from damage by ROS. A possible expla- ing that it suffers from a lack of lipid biosynthesis nation for the fact that sod-1 is necessary for cor- products. The acl1 gene is expressed mainly dur- rect fruiting-body morphology is that SOD-1 is in- ing vegetative growth prior to sexual development, volved in generating a light-dependent ROS gradi- which fits a model of the vegetative mycelium ac- entthatcontrolsneckpositioning. quiring nutrients that are mobilized later during In Podospora anserina,amutantofthetran- fruiting-body formation (Nowrousian et al. 1999, scription factor GRISEA that is involved in cellular 2000). Another mutation with implications for fatty copper homeostasis is female-sterile (Osiewacz acid metabolism in fruiting-body development is and Nuber 1996). Copper is an essential cofactor car1 of Podospora anserina.CAR1isaperoxisomal for several enzymes, among these some superoxide protein necessary for peroxisome biogenesis, and dismutases as well as cytochrome oxidase (COX), interestingly, the car1 mutant has defects in karyo- which is involved in the generation of ROS. Another gamy, and therefore is sterile (Berteaux-Lecellier mutant, in which the gene encoding a mitochon- et al. 1995). In peroxisomes, mobilization of fatty drial chaperone targeting copper to COX was acids by β-oxidation takes place, and the phenotype deleted, showed delayed perithecial formation and of the car1 mutant might be due to a disturbed fatty reduced ascus production (Stumpferl et al. 2004). acid metabolism. In contrast to the grisea mutant, this PaCox17 Higher metabolic demands of fruiting-body mutant still had superoxide dismutase activity but formation versus vegetative growth or asexual respired via an alternative oxidase pathway, which sporulation can also be inferred by analyses of might indicate that the balance between the gener- various complex I mutants of N. crassa (Videira ation and degradation of ROS is disturbed in both and Duarte 2001). Complex I of the respiratory mutants, albeit not in the same way (Stumpferl chain is a multi-subunit, proton-pumping NADH et al. 2004). Further evidence for a participation of ubiquinone oxidoreductase that is found in most ROS in fruiting-body formation comes from inves- eukaryotic mitochondria. Mutants in several com- tigation of A. nidulans.Inthisfungus,mutantsof plex I subunits are sterile in homozygous crosses. the NADPH oxidase gene, noxA,aresterile.NoxA Thismightbeduetothesubunitshavingother, generates superoxide, and is induced during sexual non-respiratory functions, but the fact that mu- development of A. nidulans. At this time, superox- tants in subunits with different functions, such as ide can be detected in Hülle cells and cleistothecia complex assembly or reductase activity, display (Lara-Ortíz et al. 2003). A similar expression pat- similar phenotypes with respect to sexual differ- tern can be observed for the catalase-peroxidase entiation indicates that probably a lack of energy gene cpeA (Scherer et al. 2002). Catalase-peroxi- due to complex I malfunction is responsible for dases are enzymes that convert ROS to harmless 338 S. Pöggeler et al. compounds, thereby protecting the cell from cursor. The short precursor has a C-terminal CaaX oxidative damage. It is conceivable that noxA and (C = cysteine, a = aliphatic, and X = any amino cpeA act in concert to generate the correct amount acid residue) motif, expected to produce a mature of self-induced oxidative stress during fruiting- pheromone with a C-terminal carboxy methyl body formation. noxA expression is dependent on isoprenylated cysteine (Fig. 16.3). The two types the MAP kinase SakA. sakA mutants show prema- of pheromone precursor genes are present in the ture sexual development, and noxA is expressed in same nucleus. In heterothallic ascomycetes, pro- sakA mutants much earlier than in the wild type (Kawasaki et al. 2002; Lara-Ortíz et al. 2003). Additional information on the involvement of nox genes in fruiting-body formation was recently gained from an investigation of P. an se rina,where amutantinthenoxA ortholog PaNox1 no longer differentiates mature fruiting bodies. Additionally, amutantinasecondmemberoftheNADPHox- idase family, PaNox2,isblockedinascosporeger- mination, and both PaNox1 and PaNox2 are required for the controlled production of superoxide as well as peroxide during sexual development (Malagnac et al. 2004). These findings indicate that the generation of ROS is tightly regulated by a signal transduction network, and is an integral part of fruiting-body formation (see also Chap. 10, this volume).

2. Pheromones In heterothallic ascomycetes, mating and subsequent fruiting-body development occur only after fusion of mycelial structures of opposite mating type. In N. crassa,diffusiblepheromoneshavebeen suggestedtobeinvolvedinthematingprocess,and tobethecauseforthedirectionalgrowthoftrichog- ynestowardthemalefertilizingcellsoftheopposite mating type (Bistis 1981, 1983). In N. crassa,this directional growth of the trichogynes did not occur when the recipient male cells harbored mutations at the mating-type locus, thus suggesting that the mating-type locus regulates the pheromone production (Bistis 1981). Pheromone precursor genes encoding two different types of pheromones have been isolated from the heterothallic filamentous ascomycetes Cryphonectria parasitica, Magnaporthe grisea, N. crassa and P. an se rina,aswellasfrom the homothallic ascomycete S. macrospora (Zhang et al. 1998; Shen et al. 1999; Pöggeler 2000; Bo- browicz et al. 2002; Coppin et al. 2005). One of the Fig. 16.3. A,B Pheromones of filamentous ascomycetes. precursor genes encodes a polypeptide containing A Sequences of predicted α-factor-like pheromones from multiple repeats of a putative pheromone se- filamentous ascomycetes. Repeats are shown in white and quence bordered by protease processing sites, and boxed in black, Kex2 processing sites (KR) in white and α boxed in gray, STE13 processing sites in black and boxed in resembles the -factor precursor gene of S. cere- gray. B Sequences of predicted a-factor-like pheromones visiae (Fig. 16.3). The other gene encodes a short from filamentous ascomycetes. The prenylation signal polypeptide similar to the S. cerevisiae a-factor pre- motifs (CaaX) are boxed in grey Fruiting Bodies in Ascomycetes 339 ductionofeitherpheromoneisdirectlycontrolled In contrast to the heterothallic mycelial by transcription factors encoded by mating-type ascomycetes N. crassa, M. grisea and C. parasitica, genes, and expression of pheromone genes seems in the homothallic S. macrospora both pheromone to occur in a mating type-specific manner (see precursor genes encoding structurally different Debuchy and Turgeon, Chap. 15, this volume; pheromones are expressed in the same mycelium Herskowitz 1989; Zhang et al. 1998; Shen et al. during the entire life cycle (Pöggeler 2000). It has 1999; Bobrowicz et al. 2002; Coppin et al. 2005). recently been demonstrated that the disruption of In contrast to all other hyphal ascomycetes, the S. macrospora ppg1 gene,encodingtheα-factor- only one pheromone precursor gene, encoding like peptide pheromone, prevents production of the a hydrophilic pheromone similar to the S. cere- peptide pheromone. However, this affects neither visiae α-factor, was identified in the homothallic vegetative growth nor fruiting-body and ascospore Aspergillus nidulans (Dyer et al. 2003). development (Mayrhofer and Pöggeler 2005). In the heterothallic N. crassa,itwasshownthat pheromone precursor genes are highly expressed under conditions that favor sexual development III. Signal Transduction Cascades (Bobrowicz et al. 2002). Interestingly, an elevated transcript level of the N. crassa mfa-1 gene en- The molecular mechanisms underlying the devel- coding the hydrophobic lipopeptide pheromone opment of fruiting bodies in ascomycetes are only was observed in 7–9 day old perithecia (Kim et al. poorly understood. However, there is increasing 2002a). evidence that the external and internal stimuli are Furthermore, the expression of the N. crassa linked to genetically programmed cellular events. pheromone genes is regulated by the endogenous Usually, protein-mediated transduction of signals circadian clock in a time-of-day specific fashion. from the cell membrane to the nucleus is respon- Both genes are repressed by RCO-1, a homolog of sible for changes in gene expression. Components the S. cerevisiae transcriptional co-repressor Tup1p supposedly involved in signal transduction path- (Bobrowicz et al. 2002). ways can be subdivided into (1) receptors that Recently, it was demonstrated that male and fe- percept the signal, (2) components that transmit male fertility of heterothallic mycelial ascomycetes the signal into the cell, and (3) nuclear transcrip- depends on interactions of pheromones with their tion factors regulating gene expression. In the specific receptors. When pheromone genes were ascomycetous yeast S. cerevisiae,keycomponents deleted, spermatia were no longer able to fertilize of the signaling cascade from the cell surface to the female partner, proving that pheromones are the nucleus have been genetically characterized. In crucial for the fertility of male spermatia (Kim et al. recent years, it has became evident that principles 2002a; Turina et al. 2003; Coppin et al. 2005). In P. elucidated in yeast are applicable also to the more anserina, the function of pheromones is restricted complex developmental programs of mycelial to fertilization, while the N. crassa lipopeptide- ascomycetes (Lengeler et al. 2000). However, pheromone gene mfa-1 has also been shown to be analysis of the N. crassa genome demonstrated involved in female sexual development, ascospore that filamentous ascomycetes encode classes of production, and vegetative growth of both mating sensing molecules not found in S. cerevisiae, types (Kim et al. 2002a; Coppin et al. 2005). suggesting that alternative signaling cascades are Similarly, in C. parasitica, deletion of only one involved in development processes of mycelial of the two copies of CaaX-type pheromone genes ascomycetes (Borkovich et al. 2004). (Mf2-2) was sufficient to prevent female fertility (Zhang et al. 1993). It was therefore speculated that Mf2-2 of C. parasitica is required for a develop- A. Perception of Environmental mental phase after fertilization, and that the CaaX- and Endogenous Signals type pheromone acts in a dosage-specific manner in post-fertilization events (Turina et al. 2003). Central to many signaling pathways in eukaryotes Kim et al. (2002a) postulated an additional role is a cell surface receptor, which percepts an external for CaaX-type pheromones, in “conglutination” in chemical stimulus. Among the best characterized perithecial development, or in the cementation of signaling systems are those mediated by guanine hyphae to stabilize the sclerotial structure of the nucleotide binding (G)-protein-coupled seven- maturing perithecium. transmembrane-spanning receptors (GPCRs; 340 S. Pöggeler et al.

Neves et al. 2002). Typically, a ligand-bound resulted in the production of a few small cleis- GPCR activates or inhibits heterotrimeric G tothecia carrying a reduced number of ascospores. proteins, and transmits the signal to downstream Under homothallic conditions, an A. nidulans effectorsincludingadenylylcyclasesandprotein double-receptor-knockout strain ∆gprA/∆gprB kinases. Genome sequence analysis has shown was completely abolished in fruiting-body and that N. crassa possesses at least 10 predicted ascospore formation. Interestingly, outcrossing seven-transmembrane helix proteins that are of A. nidulans receptor-mutant strains (∆gprA/ potential G-PRCs (Galagan et al. 2003; Borkovich ∆gprB×∆gprA/∆gprB)resultedinfruiting-body et al. 2004). Three of these were characterized and ascospore formation at wild-type level, at the molecular level. The nop-1 gene encodes suggesting that in A. nidulans the pheromone a seven-transmembrane helix retinal-binding pro- receptors GprA/B are specifically required for self- tein homologous to archaeal rhodopsins. Analysis fertilization, and not for sexual development per se of nop-1 deletion strains did not reveal obvious de- (Seo et al. 2004). By contrast, deletion of the GPRD- fects in light-regulated processes or fruiting-body encoding gprD gene causes extremely restricted development under normal laboratory conditions hyphal growth, delayed conidial germination, and (Bieszke et al. 1999). uncontrolled activation of sexual development. Two genes, designated pre-1 and pre-2,encod- Since elimination of sexual development rescues ing putative pheromone receptor genes similar to both growth and developmental abnormalities in S. cerevisiae a-factor receptor (Ste3p) and α-factor ∆gprD strains, it was suggested that the primary receptor (Ste2p), respectively, have been identified role of GprD is to negatively regulate sexual in N. crassa and S. macrospora (Pöggeler and Kück development (Han et al. 2004). Moreover, it was 2001). Using a heterologous yeast system, it has demonstrated that deletion of pheromone receptor been shown that the S. macrospora receptor PRE2 genes gprA and or gprB suppressed growth defects facilitates all aspects of the yeast pheromone re- caused by the deletion of the gprD gene. This result sponse in S. cerevisiae MATa cells lacking the Ste2p implies that pheromone receptors GprA and GprB receptor, when activated by the S. macrospora pep- function downstream of GprD-mediated negative tide pheromone. Therefore, one may conclude that control of sexual development (Seo et al. 2004). the receptor encoded by the pre2 gene functions As has been described in the section above, as a GPCR in S. macrospora,too(Mayrhoferand fruiting-body development in filamentous asco- Pöggeler 2005). mycetesisinfluencedbyavarietyofenviron- Northern and reverse transcription-poly- mental stimuli and endogenous factors. In N. merasechainreactionanalysesindicatethatinthe crassa, known elements in light sensing involved heterothallic N. crassa,incontrasttopheromone in fruiting-body development include the white precursorgenes,expressionofthereceptorgenes collar complex, which acts as a blue-light photore- does not occur in a mating type-specific manner ceptor as well as a transcription factor to regulate (Pöggeler and Kück 2001; Kim and Borkovich transcription of target genes in a light-dependent 2004). Recently, Kim and Borkovich (2004) demon- manner (see Sect. II.A.2, and Corrochano and strated that deletion of the N. crassa pre-1 gene Galland, Chap. 13, this volume). In addition to does not affect vegetative growth or male fertility. these two proteins, numerous other putative However, protoperithecia from ∆pre1 mat A light-sensing genes have been identified in the mutants were shown to be female-sterile, because genomic sequence of N. crassa,buttheirrole their trichogynes are unable to recognize and fuse in fruiting-body development has still to be with mat a cells. In the genome of the homothallic elucidated (Borkovich et al. 2004). A. nidulans,ninegenes(gprA-gprI)forpotential Other environmental signals influencing seven-transmembrane spanning G-PRCs have fruiting-body development, such as osmolarity, been identified. Six of nine putative GPCRs have nutrient levels, oxygen levels, and cellular redox been disrupted and three genes, gprA, gprB and status, might be sensed by two-component signal gprD, were found to play a central role in coor- transduction pathways. dinating hyphal growth and sexual development Two-component regulatory systems are com- (Han et al. 2004; Seo et al. 2004). Putative G-PRCs posed of an autophosphorylating sensor histidine similar to the S. cerevisiae pheromone receptors kinase and a response regulator. In contrast to Ste2pandSte3pwereshowntobeencodedbygprA yeasts, filamentous ascomycetes encode an exten- and gprB, respectively. Deletion of gprA or gprB sive family of two-component signaling proteins Fruiting Bodies in Ascomycetes 341

(Catlett et al. 2003). Eleven genes encoding puta- B. Signal Transduction Pathways tive histidine kinases have been identified in the N. crassa genome. So far, only two of them, nik- In response to extracellular stimuli, two cyto- 1/os-1 and nik-2, have been characterized, but seem plasmic signaling branches defined by cAMP- not to be involved in fruiting-body development. dependent protein kinases (PKA) and mitogen- Whereas NIK-1 is an osmosensing histidine kinase activated protein kinases regulate gene expression that plays an important role in the regulation of that finally leads to ascocarp formation (Lengeler cell wall assembly and cell responses to changes et al. 2000). Upstream of these two signaling in external osmolarity, nik-2 deletion mutants ex- cascades, either heterotrimeric G proteins or ras hibit no obvious phenotypes (Alex et al. 1996; Schu- and ras-like proteins act to relay extracellular macher et al. 1997; Borkovich et al. 2004). Similarly, ligand-stimulated signals to the cytoplasm. In two histidine kinases analyzed in A. nidulans were addition to these four classes of proteins, several eithershowntobeinvolvedintheformationof highly conserved proteins of unknown function asexual spores, or disruption yielded no obvious have been identified by complementation of clues to their function (Virginia et al. 2000; Fu- fungal developmental mutants (Fig. 16.4). These rukawa et al. 2002). The involvement of the other proteins are supposedly part of signal transduc- two-component systems in ascocarp development tion pathways that lead to ascocarp formation awaits testing. (Table 16.1).

Fig. 16.4. Signal transduction pathways leading to fruiting-body development in ﬁlamen- tous ascomycetes 342 S. Pöggeler et al.

1. Heterotrimeric G Proteins cial development (Gao and Nuss 1996). Mutation of the Gα subunit gene cga-1 of the maize pathogen Upon activation of G-PRC receptors, hetero- Cochliobolus heterostrophus leads to defects in sev- trimeric G proteins catalyze the exchange of GDP eral developmental pathways. Conidia from cga-1 for GTP on the G protein α subunit, which leads to mutants germinate as abnormal, straight-growing its dissociation from the βγ subunits. Either Gα,or germ tubes that form few appressoria, and the mu- Gβγ, or both are then free to activate downstream tants are female-sterile (Horwitz et al. 1999; Degani effectors (Dohlman 2002). In N. crassa and A. et al. 2004). In A. nidulans,theGα subunit FadA, nidulans as well as other filamentous ascomycetes, along with the Gβ subunit SfdA, was shown to be an it was demonstrated that subunits of G proteins active participant in signaling pathways that gov- are important for hyphal growth, conidiation, ern critical decisions in the ascocarp development. and fruiting-body development (Table 16.1). The A. nidulans strains carrying a deletion of the fadA genome of N. crassa contains three genes (gna-1, gene fail to form cleistothecia. By contrast, strains gna-2 and gna-3)encodingGα subunits, a single carrying a dominant-negative mutation in the fadA gene encoding the Gβ (gnb-1)subunit,andasingle (fadAG203R)genedisplayanincreaseinHüllecell gene encoding the Gγ (gng-1)subunit(Borkovich formation but no cleistothecia formation. A similar et al. 2004). During the sexual cycle, GNA-1 is phenotype was observed when the sfaD gene was necessary for female fertility, whereas GNA-3, deleted (Rosén et al. 1999). In addition to FadA, which is also important for asexual sporulation, FlbA, a member of the regulator of G protein sig- mediates ascospore maturation (Ivey et al. 1996; nalling (RGS) proteins that function as GTPase ac- Yang and Borkovich 1999; Kays et al. 2000). tivating proteins, was shown to be involved in cleis- Loss of gna-2 has no obvious effect on N. crassa tothecia formation in A. nidulans (Han et al. 2001). growth and development; however, the double ∆ ∆ mutant gna-1/ gna-2 was shown to have more 2. RAS and RAS-Like Proteins pronounced defects in female fertility than was the case for ∆gna-1 strains. This suggests that RAS and RAS-like proteins are small GTP-binding gna-1 and gna-2 have overlapping functions in proteins, which reside on the inner surface of the sexual development (Baasiri et al. 1997; Kays and plasma membrane. They are able to transduce sig- Borkovich 2004). ∆gnb-1 and ∆gng-1 strains of nalstothecytoplasmicsignalingcascades,andcon- N. crassa are able to function as males during trol a variety of essential cellular processes (Vojtek crosses with wild-type strains. When used as and Der 1998). female parents, however, protoperithecia do not Small GTP-binding proteins are generally develop normally after fertilization, and they grouped into five subfamilies named after their produce only small perithecia with aberrant prototypical member: RAS, RHO, RAN, RAB, fertilized reproductive structures (Yanget al. 2002; and ARF. The RAS superfamily of GTP-binding Krystofova and Borkovich 2005). proteins comprises over 100 members, which act as In the closely related pyrenomycete Podospora molecular switches by cycling between an inactive anserina,themod-D gene encoding a Gα sub- GDP-bound state and an active GTP-bound state unit and cloned as a suppressor of nonallelic (Takai et al. 2001). heterokaryon incompatibility was shown to The pyrenomycete N. crassa encodes two RAS be involved in protoperithecial development proteins and three RAS-like proteins (Borkovich (Loubradou et al. 1999). et al. 2004). To date, an involvement in ascocarp Similar to N. crassa,thericepathogenMagna- development has been demonstrated only for the porthe grisea encodes three Gα subunits. Female RAS-like N. crassa protein KREV-1, a homolog of fertility is mediated by MAG-B, whereas the other the mammalian RAP protein, and the S. cerevisiae two subunits MAG-A and MAG-C are required for Rsp1p protein. Disruption of the N. crassa krev-1 the production of mature asci (Liu and Dean 1997; gene by RIP was not correlated with growth ab- Fang and Dean 2000). Targeted disruption of two normalities, but mutants overexpressing a consti- Gα subunitgenesinthechestnutblightfungusCry- tutively active KREV-1 were shown to have defects phonectria parasitica revealed roles for the subunit in the sexual cycle. Mutations that are thought to CPG-1 in fungal reproduction, virulence, and veg- locktheKREV-1proteinintotheactiveGTP-bound etative growth. Disruption of the second Gα sub- form completely inhibit the development of pro- unit gene, cpg-2, was found to enhance perithe- toperithecia into perithecia, whereas a dominant Fruiting Bodies in Ascomycetes 343 negative mutation of KREV-1 resulted in some en- The involvement of catalytic and regulatory largement of protoperithecia (Ito et al. 1997). subunits of PKA has so far been demonstrated only In A. nidulans, mutations inthe rasA gene cause with respect to the asexual development, hyphal aberrations in conidial germination and asexual growth, and pathogenicity of N. crassa, A. nidu- development, but the role of RAS- and RAS-like lans and M. grisea, respectively (Mitchell and Dean proteins in cleistothecium formation has not yet 1995; Bruno et al. 1996; Fillinger et al. 2002). How- been determined (Som and Kolaparthi 1994; Os- ever, effects of PKA on fruiting-body formation herov and May 2000; Fillinger et al. 2002). have not yet been analyzed. Due to the presence of genes in the N. crassa genome encoding G-PRCs 3. cAMP Activated Protein Kinases PKA similar to slime mold cAMP receptors, it was recently suggested that cAMP may also serve as an Activated RAS, RAS-like proteins, Gα or Gβγ environmental signal and G-PRC ligand inN. crassa subunits can regulate downstream effectors such (Borkovich et al. 2004). as adenylyl cyclase and mitogen-activated protein kinase (MAPK) cascades (see Sect. III.B.4). 4. Mitogen-Activated Protein Kinase (MAPK) Adenylyl cyclase is a membrane-bound enzyme Pathways that produces cyclic AMP (cAMP) from ATP. cAMP is a ubiquitous secondary messenger in Mitogen-activated protein kinase (MAPK) path- prokaryotic and eukaryotic cells. In filamentous ways regulate eukaryotic gene expression in re- ascomycetes, cAMP signaling is involved in such sponse to extracellular stimuli. The basic assembly diversecellularprocessesasstressresponse, of MAPK pathways is a three-component module metabolism, pathogenicity, and sexual develop- conserved from yeast to humans. ment (Kronstad et al. 1998; Lengeler et al. 2000). The MAPK module includes three kinases that A well-characterized intracellular target of cAMP establish a sequential activation pathway compris- is the regulatory subunit of protein kinase A (PKA). ing a MAPK kinase kinase (MAPKKK), MAPK ki- PKA is a tetrameric enzyme that is composed of nase (MAPKK), and MAPK (Widmann et al. 1999). two regulatory subunits and two catalytic subunits. MAPK is activated by phosphorylation on con- BindingofcAMPtotheregulatorysubunitsreleases served tyrosine and threonine residues by MAKK. the catalytic subunits. The latter phosporylate tar- In turn, MAPKK is activated by phosphorylation on get proteins involved in cAMP-regulated processes conserved serine and threonine residues by MAP- (Dickman and Yarden 1999; Taylor et al. 2004). KKK. MAPKKK become activated by phosphory- In the plant pathogen M. grisea,itwasshown lation in response to various extracellular stimuli. that the sexual cycle is dependent on cAMP. M. The activated MAPK can translocate to the nucleus grisea mutants lacking the adenylyl cyclase gene where it phosphorylates, and thus activates tran- mac-1 are female-sterile, and mate only when ex- scription factors (Dickman and Yarden 1999). ogenous cAMP is supplied (Choi and Dean 1997; In the filamentous ascomycete N. crassa, Adachi and Hamer 1998). By contrast, the N. crassa three MAPK modules have been identified by cr-1 mutant, lacking adenylyl cyclase activity and genome sequence analysis (Borkovich et al. cAMP,isabletofunctionasmaleandfemalepart- 2004). These correspond to those for pheromone ner in sexual crosses, but exhibits delayed perithe- response/filamentation, osmosensing/stress and cial and ascospore production, compared to the cell integrity pathways in the yeasts S. pombe wild type (Perkins et al. 1982; Ivey et al. 2002). and S. cerevisiae. Three different MAPKs and Female sterility of the cr-1 mutant was observed two different MAPKKKs have been shown to be only in a ∆gna-1 background. Unlike the ∆gna-1 involved in fruiting-body development in different mutant, the ∆gna-1/∆cr-1 double mutant does not mycelial ascomycetes (Table 16.1). Mutation of form protoperithecia, suggesting that cAMP may the corresponding genes always leads to multiple be required for protoperithecial development. A re- phenotypic defects, including defects in ascocarp cent study explored the contribution of all three Gα formation. Deletion of the N. crassa mak-2 gene subunits on the cAMP level. The effects of mutating encoding a Fus3p MAPK was shown to result in gna-1 and gna-3 were shown to be additive with re- loss of hyphal fusion and female sterility (Pandey spect to the adenylyl cyclase activity, whereas loss et al. 2004; Li et al. 2005). of gna-2 did not appreciably affect adenylyl cyclase The MAPK Hog1p plays an essential role in os- activity (Kays and Borkovich 2004). motic stress signaling and osmoadaptation in the 344 S. Pöggeler et al. yeast S. cerevisiae (Hohmann 2002). In A. nidulans, ally induced by exposure to pheromone, and the Hog1p homolog SakA/HogA was demonstrated to participate in the signaling pathway leading to be involved not only in stress signal transduc- to pheromone-mediated G1 arrest (Kemp and tion but also in sexual development. A. nidulans Sprague 2003). ∆sakA mutants display premature sexual develop- Similarly, in the related pyrenomycete Po- ment, and produce two times more cleistothecia at dospora anserina, there is evidence for a link least 24 h earlier than wild-type strains (Kawasaki between genes involved in incompatibility and et al. 2002). In the plant pathogens M. grisea and perithecial development. A screen for suppressors Fusarium graminearum, it was demonstrated that of vegetative incompatibility led to the isolation homologs of the S. cerevisiae Slt2p MAPK are re- of the P. anserina mod-A gene, which is not quired for plant infection, and also for female fer- only responsible for growth arrest in the self- tility (Xu et al. 1998; Hou et al. 2002). incompatible strains, but also involved in the The MAPKKK NRC-1 of N. crassa functions controlofthedevelopmentoffemaleorgans.The to repress the onset of conidiation and is required MODA-encoded polypeptide is rich in proline for female fertility, because a N. crassa nrc-1 mu- residues, which are clustered in a domain contain- tant is unable to make protoperithecia (Kothe and ing a motif that displays similarity to SH3-binding Free 1998). It was recently demonstrated by Pandey motifs (Barreau et al. 1998). et al. (2004) that an nrc-1 mutant shares many of the In the homothallic pyrenomycete Sordaria same phenotypic traits as the mak-2 mutant, and is macrospora,thesterilemutantpro11carriesade- also a hyphal fusion mutant, implying that NRC-1 fect in the pro11 gene encoding a multimodular acts upstream of MAK-2 during fruiting-body de- WD40-repeat protein. PRO11 shows significant velopment. Similarly to the N. crassa NRC-1, the A. homology to several vertebrate WD40 proteins, nidulans SteC regulates conidiophore development such as striatin or zinedin, which seem to be and is essential for cleistothecial development (Wei involved in Ca2+-dependent signaling in cells of et al. 2003). the central nervous system, and supposedly function as scaffolding proteins linking signaling and 5. Other Putative Signaling Proteins eukaryotic endocytosis. Most importantly, a cDNA encoding the mouse striatin caused a functional In addition to the components of conventional substitution of the S. macrospora wild-type gene signal transduction pathways, forward genetic with a restoration of fertility in the pro11 mutant, screens in filamentous ascomycetes identified suggesting that an evolutionary conserved cellular several conserved proteins that may play key roles process in eukaryotic cell differentiation may in fruiting-body development, but have so far not regulate fruiting-body formation (Pöggeler and been shown to be involved in signal transduction Kück 2004). processes in other eukaryotes. During a screen for suppressors of vegetative incompatibility in N. crassa, Xiang et al. (2002) isolated a deletion C. Transcription Factors mutant that displayed pleiotropic defects including suppression of vegetative incompatibility, altered From the preceding section, it is evident that di- conidiation pattern, lack of hyphal fusion, and verse signal transduction pathways interpret en- female sterility. A single gene termed ham-2 vironmental or intrinsic signals. Eventually, signal was shown to complement hyphal fusion and transduction initiates morphogenesis by activating female fertility, but not vegetative incompati- transcription factors that in turn activate or repress bility and condiation pattern. The ham-2 gene cell- or tissue-specific expression of morphogenetic encodes a putative transmembrane protein that genes. Most transcription factors involved in asco- is highly conserved, but of unknown function carp formation have been identified in A. nidulans among eukaryotes. Since the ham-2 mutant forms (Table 16.1). Some of these are thought to be direct only few protoperithecial-like structures, it was targets of signaling pathways. speculated that a HAM-2-dependent hyphal In A. nidulans,SteA,ahomologoftheS. cere- fusion process may be required for the formation visiae homeodomain transcription factor Ste12p, of female reproductive structures (Xiang et al. wasisolatedandshowntoberequiredforcleis- 2002). In addition, the S. cerevisiae homolog of tothecia formation. In the budding yeast, Ste12p HAM-2, Far11p, was shown to be transcription- plays a key role in coupling signal transduction Fruiting Bodies in Ascomycetes 345 through MAP kinase modules to cell-specific or of different classes from hyphal ascomycetes have morphogenesis-specific gene expression required been identified that regulate fruiting-body forma- for mating and pseudohyphal filamentous growth. tion. Amongst these is the C6 zinc finger transcrip- An A. nidulans ∆steA strain is sterile and differen- tion factor PRO1, isolated in a forward genetic tiates neither ascogenous hyphae nor cleistothecia. screen from the homothallic pyrenomycete Sor- However, the development of sexual cycle-specific daria macrospora.Thedevelopmentalmutantpro1 Hülle cells and asexual conidiation is unaffected forms only protoperithecia, and is unable to per- (Vallim et al. 2000). A similar phenotype has been form the transition into mature perithecia (Masloff observed in N. crassa. pp-1 mutants of N. crassa fail et al. 1999). Functional analysis of the PRO1 tran- to develop protoperithecia. In addition, ascospores scription factor revealed that the Zn(II)2Cys6 bi- carrying null mutations of the pp-1 gene are non- nuclear cluster is a prerequisite for the develop- viable (Li et al. 2005). mental function of the protein. Fertility of the S. In a forward genetic screen of A. nidulans,Han macrospora pro1 mutant can be restored by the et al. (2001) isolated the nsdD (never in sexual pro1 homolog from N. crassa (Masloff et al. 2002). development) gene encoding a GATA-type tran- Recently, it was shown that the putative Zn(II)2Cys6 scription factor. Deletion of nsdD resulted in loss transcription factor RosA from A. nidulans,shar- of cleistothecia or Hülle cell formation, even un- ing 38% sequence similarity to the S. macrospora der conditions that preferentially promote sexual PRO1,isanegativeregulatorofsexualdevelopment development, indicating that NsdD is necessary in A. nidulans (Vienken et al. 2004). forascocarpformation.TranscriptionfactorsStuA, Besides the transcription factors described MedA, and DopA are also required during sexual above, mating type-encoded transcription factors reproduction in A. nidulans.Whereas∆stuA and arealsoinvolvedinfruiting-bodydevelopmentof ∆dopA strains fail to differentiate Hülle cells and filamentous ascomycetes (reviewed by Debuchy cleistothecia, ∆medA strains form extensive masses and Turgeon, Chap. 15, this volume). Mating pro- of unorganized Hülle cells, but fail to differentiate teins, as master regulatory transcription factors, cleistothecia. control pathways of cell speciation as well as sexual In contrast to steA and nsdD, stuA, medA as well morphogenesis in heterothallic and homothallic as dopA are developmental modifier genes that are ascomycetes. They are not only necessary for also involved in asexual development of A. nidu- fertilization, but are also required for subsequent lans (Busby et al. 1996; Dutton et al. 1997; Wu and development of the fertilized pre-fruiting bodies Miller 1997; Pascon Castiglioni and Miller 2000). (Coppin et al. 1997; Pöggeler 2001). The N. crassa homolog of the A. nidulans stuA gene, asm-1, has also been shown to be involved in sexual development, since deletion of asm-1 de- stroys the ability to make protoperithecia, but does IV. Structural Components Involved not affect male-specific functions (Aramayo et al. in Fruiting-Body Development 1996). So far, information about the sexual regulatory Fruiting-body formation involves the differentia- network in A. nidulans is limited. However, Val- tion of many morphologically distinct cell types. lim et al. (2000) reported that steA lies upstream of Besides the cells that participate directly in karyo- medAin the same regulatory pathway, and that SteA gamyandmeiosis,manymorespecializedcelltypes directly or indirectly represses medA transcription are formed that comprise the mature fruiting body. (see Fischer and Kües, Chap. 14, this volume). Up- Of the 28 recognized cell types of N. crassa,15 regulation of nsdD resulted in the production of occur only during fruiting-body formation (Bis- barren cleistothecia in the ∆gprA/∆gprB double tis et al. 2003). As the cell wall is the main form- mutant.ThisresultsuggeststhatNsdDcanpar- giving structure of the fungal cell, it can be ex- tially rescue the developmental defects caused by pected that enzymes involved in cell wall biogene- the deletion of GPCRs, and that GprA/B-mediated sis and metabolism are required and coordinately signaling may activate other genes necessary for the regulated during sexual development. In addition, maturation of cleistothecia (Seo et al. 2004). In turn, genes for cytoskeleton structure and organization nsdD expression was shown to be under control of have in some cases been shown to be specifically in- FadA-mediated signaling (Han et al. 2001). As sum- volvedinsexualdevelopment,andtheseandthecell marized in Table 16.1, other transcription factors wall-related genes will be discussed in this section. 346 S. Pöggeler et al.

A. The Cytoskeleton in Fruiting-Body during asexual sporulation, and chsB during all Development stages of growth and development, whereas chsC was expressed during early vegetative growth as Genes encoding components or regulators of the well as sexual development. In the latter case, chsC cytoskeleton have been shown to contribute to sex- expression was restricted to cleistothecia and as- ual development in A. nidulans and P. an se rina. A. cospores (Lee et al. 2004). In T. borchii,expression nidulans has two genes encoding α-tubulin, tubA of three genes for chitin synthases was analyzed and tubB.WhereastubA is necessary for mitosis in vegetative hyphae and fruiting bodies. All and nuclear migration, tubB is essential for as- three were constitutively expressed in vegetative cospore formation (Kirk and Morris 1991). In P. mycelium, but two of them were additionally ex- anserina,itwasshownthatanami1mutantismale- pressed in fruiting bodies. One of these was found sterile and displays delayed fruiting-body forma- in sporogenic tissue, and the other in the vegetative tion (Bouhouche et al. 2004). ami1 encodes a ho- tissue of the fruiting body (Balestrini et al. 2000). molog of the A. nidulans apsA gene, which is a pro- Theseexamplesindicatethat,mostlikely,chitin tein necessary for nuclear positioning, most likely synthases have specific but overlapping functions by regulating components of the dynein pathway. during different stages of fungal development. Thus, the cytoskeleton-related genes involved in sexual development have so far turned out to be important for nuclear migration and cell cycle events. 2. Carbohydrates After chitin, carbohydrates are another important B. TheCellWallinFruiting-BodyDevelopment constituent of the fungal cell wall, one main component being β-1,3-glucan (Walser et al. 2003). Apart The function of fruiting bodies is the protection from being structural elements, it has been pro- and discharge of the ascospores, and this is posed that carbohydrates are stored during vegeta- achieved by developing morphologically distinct tive growth to be mobilized as a carbon source for structures that are often characterized by cells sexual development. One of these potential storage with rigid and heavily pigmented cell walls. Thus, carbohydrates is α-1,3-glucan. It was shown to ac- it has long been proposed that enzymes involved in cumulate during vegetative growth of A. nidulans, cell wall biogenesis and metabolism are essential and is degraded at the onset of sexual development for sexual development in ascomycetes. Analyses (Zonneveld 1972). This finding correlates well with have concentrated on several classes of genes thefactthatthegeneforα-1,3-glucanase, mutA,is considered to have a role in development, namely, expressedduringsexualdevelopmentanddegrades genes involved in pigmentation, chitin synthases, α-1,3-glucan to yield glucose. lectins, hydrophobins, and genes involved in Interestingly, MUTA is mainly found in Hülle carbohydrate metabolism of the cell wall. Some of cells whereas α-1,3-glucan is located mostly in the theresultsaredescribedbelow(seealsoChaps.4 walls of cells other than Hülle cells. This might and 5, this volume). indicatethatHüllecellsproduceglucanase,which then degrades glucan in the cell walls of storage 1. Chitin hyphae to yield carbohydrates that can be absorbed by the still intact Hülle cells (Zonneveld 1972; Wei One of the characteristic components of the fungal et al. 2001). cell wall is chitin, and central enzymes in chitin However, mutA mutants are still able to form biosynthesis are chitin synthases. Fungi usually fruiting bodies, which indicates that other forms possess several chitin synthase genes, e.g., the of carbohydrates can be used during fruiting-body genome of N. crassa contains seven chitin synthase development (Wei et al. 2001). One of these might genes (Borkovich et al. 2004). Chitin synthases that be rhamnogalacturonan, as it has been shown that might be specifically required for fruiting-body a putative rhamnogalacturonase encoded by the formation have not been identified with certainty. asd-1 gene is essential for ascospore formation in N. However, chitin synthase genes that are expressed crassa (Nelson and Metzenberg 1992; Nelson et al. preferentially in fruiting-body tissue have been 1997b). It is not yet clear whether rhamnogalactur- found in A. nidulans and T. borchii.InA. nidulans, onan inhibits sexual development and is degraded the three chitin synthase genes chsA, chsB and chsC by ASD-1, or if the rhamnogalacturonan degrada- have been investigated. chsA was expressed solely tion products are needed for development. Fruiting Bodies in Ascomycetes 347

The fact that fruiting-body formation requires tant with white perithecia (Graziani et al. 2004). high amounts of energy in the form of carbohy- The mutant, however, is fertile. Therefore, the lack drates initially stored in vegetative hyphae, and of perithecial pigmentation does not preclude as- subsequently released mostly in the extracellular cospore maturation (Babai-Ahary et al. 1982), al- space, would imply the existence of effective trans- though it might be interesting to determine perfor- port systems for nutrient uptake into the cells of manceofthemutantstrainundernaturalgrowth the developing fruiting body. One such transporter conditions. for hexoses that is localized in ascogenous hyphae was described in A. nidulans (Wei et al. 2004). 4. Cell Wall Proteins The gene for this transporter, hxtA, is not essential for sexual development, but as there are at In addition to carbohydrates and pigment least 17 putative hexose transporter genes in the molecules, proteins comprise a sizeable part A. nidulans genome, this might be due to the exis- of the fungal cell wall. Two classes of proteins, tence of functionally redundant proteins (Wei et al. hydrophobins and lectins, have been characterized 2004). extensively in higher basidiomycetes where they are implied in mushroom formation (see also 3. Pigments Chap. 19, this volume). Hydrophobins can self- assemble at water/air interfaces, and form highly Other common components of fungal cell walls insoluble amphipathic films. These films can are pigments, e.g., brown or black melanins. attach to the hydrophilic cellular surfaces, thereby Melanins stabilize the cell wall and offer protection orienting the hydrophobic side to the outside, against UV light-induced DNA damage, but also which allows the fungus to break a water/air have additional functions. For example, they are interface and grow into the air (Wösten et al. necessary for appressorial penetration of plant 1999). Lectins are carbohydrate-binding, mostly tissue in several fungal plant pathogens, and extracellular proteins that occur in virtually all for pathogenicity of several human pathogens classes of organisms. They have been implicated (Howard and Valent 1996; Perpetua et al. 1996; intheinteractionoffungiwithotherorganismsas Jacobson 2000). Two biochemical pathways have well as fruiting-body formation in basidiomycetes been recognized for fungal melanin production, (Walser et al. 2003). Genes encoding hydrophobins the DHN (dihydroxynaphtalene)- and the DOPA and lectins have been found in ascomycetes, too, (dihydroxyphenylalanine)-melanin biosynthesis but whether any of these play a role in sexual pathways (Langfelder et al. 2003). Both pathways development is not yet clear. contain laccases for the first (DOPA) or last Hydrophobins from N. crassa, A. nidulans and (DHN) biosynthetic step, respectively. The DHN M. grisea havebeenshowntobeinvolvedinthefor- pathway involves a polyketide synthase in its initial mationofthewater-repellentcoatofconidiospores, step, but laccases and polyketide synthases also but not in fruiting-body development (Stringer participate in a variety of biosynthetic pathways et al. 1991; Bell-Pedersen et al. 1992; Stringer and not necessarily involved in pigment formation. Timberlake 1995; Talbot et al. 1996). Laccase and polyketide synthase genes have been As fungi can contain several members of the identified in several fungi, and have been implied hydrophobin family in their genomes (Segers et al. in fruiting-body pigmentation of ascomycetes, but 1999; Fuchs et al. 2004), addressing the question so far evidence for a genetic control of melanin whether any of these are involved in fruiting-body biosynthesis in black or darkly pigmented fruiting morphogenesis might be a complex task involving bodies is missing. the generation of mutants in more than one hy- It was shown for A. nidulans that laccase activ- drophobin gene. Similarly to hydrophobins, lectins ity is located in young cleistothecia and Hülle cells, are thought to be involved in fruiting-body for- and that a sterile mutant had reduced laccase activ- mation, but a requirement for lectins has yet to ity, but the corresponding laccase gene(s) have not beshowninanyfungus.Theonlydescribedmu- yet been identified (Kurtz and Champe 1981; Her- tant in a lectin-encoding gene of an ascomycete is mann et al. 1983). In the plant pathogen Nectria the aol mutant of Arthrobotrys oligospora (Balogh haematococca,apolyketidesynthase,PKSN,was et al. 2003), but as no perfect stage of A. oligospora showntobeessentialforthesynthesisofthered is known, the question of an involvement of AOL perithecial pigment by complementation of a mu- in sexual development cannot be addressed. 348 S. Pöggeler et al.

5. AreMultipleGeneswithOverlappingFunctions way is obvious that predominantly directs fruit- Involved in Cell Wall Metabolism? ing-body development in ascomycetes. In summary, it has become evident that multi- The information about the molecular and genetic cellular ascocarp development requires precise in- basis of cell wall metabolism during fruiting-body tegration of a number of fundamental biological formationinascomycetesisratherlimited.Arecur- processes. The sexual pathway in ascomycetes pro- ringthemearemutantsin“candidategenes”that vides a valuable experimental system for studying surprisingly do not have any discernible pheno- the mechanism and regulation of developmental type (see Sect. IV.B.2–4). Especially with the avail- processes that are usually more complex in ani- ability of whole genome sequences, it has become mal and plant development. The analysis of fun- increasingly clear that one of the reasons for these gal genomes is a starting point for further under- ﬁndings might be the existence of large gene fam- standing developmental processes in mycelial fungi ilies most likely with partly overlapping functions. and other multicellular eukaryotes. Post-genomic This does seem to be the case for most of the genes analysis, including transcriptome and proteome involved in the biosynthesis of cell wall compo- assessments, will hopefully decipher components nents. A possible explanation for this might be directing multicellular differentiation processes in the fact that the cell wall is a highly important ascomycetes, and ﬁnally as a long-term objective structure for fungi – it is form-giving as well as will help to understand eukaryotic differentiation protecting – but makes assessments of single com- processes at the molecular level. ponents somewhat tedious. However, analysis of whole genome data, especially cross-species com- Acknowledgements.WethankDr.S.MasloffandDr.S.Risch parisons as well as large-scale expression studies, for microscopic images. The work of the authors is funded could help to identify candidates for future gene by the ‘Deutsche Forschungsgemeinschaft’ (DFG). inactivation projects. References

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