Morphogenesis and vegetative differentiation in

J. Genet., Vol. 75, Number 3, December 1996, pp. 361-374. (() Indian Academy of Sciences

Morphogenesis and vegetative differentiation in filamentous fungi

DANIEL J. EBBOLE Program for the Biology of Filamentous Fungi, Texas A&M University, College Station, TX 77843-2132, USA

Abstract. Filamentous fungi are a morphologically diverse group of organisms. Growth occurs by extension of the hyphal tip. Cell shape is primarily determined by the location and rate of cell wall deposition. Signalling pathways that control these processes result in the formation of distinct cell types and multicellular structures. Examples of eonidiation and appressorium tbrmation in several filamentous ascomycctes suggest that some common features exist in the regulatory mechanisms governing asexual development in these fungi.

Keywords. Saccharomyces cerevisiae; Netlrospora; Aspergillus; Magnaporthe; signal transduction; plant pathogen; growth polarity.

1. Introduction

Growth polarity, hyphal branching and orientation of tip growth are the building blocks of fungal morphogenesis. How these basic processes are coordinated to produce the microscopic and macroscopic rnorphological diversity observed in filamentous fungi is largely unknown. The understanding of morphogenesis in Saccharomyces cerevisiae is most impressive because of the integration of information concerning physiology, genetics and cell biology (Cid 1995). S. cerevisiae is relatively unsophisticated in its development; however, it can alter its growth mode to produce elongated diploid cells (pseudohyphal growth) and haploid cells that are capable of invasive growth (Herskowitz 1995). The signal transduction pathways controlling differentiation may be similar for all fungi. The sharing of components in the signal transduction pathways for sexual and asexual development in yeast makes it likely that MAP (mitogen-activated protein) kinase pathways will prove to be critical in rnorphogenesis in the higher fnngi (Herskowitz 1995). It is only recently that homologous components of signal transduction pathways, such as kinases (Mitchell and Dean 1995; Alex et al. 1996; Xu and Hamer 1996) and G proteins (Turner and Borkovich 1993; Chen et al. 1996), have been cloned to examine their functions in filamentous fungi.

Examples from a number of filamentous fungi demonstrate that the building- block processes can be studied genetically. In Neurospora crassa, for example, the Tncb (microcycle blastoconidiation; Maheshwari 1991) gene is required to establish growth polarity and was recently shown to encode the regulatory subunit of cAMP- dependent protein kinase (Bruno et aI. 1996). N. crassa cot-1 encodes a kinase that appears to regulate both growth polarity and branching (Yarden et al. 1992). Even the analysis of orientation of hyphal tip growth is amenable to genetic analysis. N. crassa ropy (to) mutants display exaggerated curling ofhyphae. In these mutants, in addition to altered orientation of tip growth, the distribution of nuclei is altered. It was shown that ro genes encode subunits of the cytoplasmic dynein and dynactin complexes

361

362 Daniel J. Ebbote

(Plamann et al. 1994). However, integration of these types of studies is needed to define the basic pathways that control the building blocks of fungal morphogenesis.

Current genetic studies of developmental processes in filamentous fungi can be viewed as an examination of how the building-block functions are coordinated with respect to a temporal sequence, rather than an examination of the building blocks themselves. It is easiest to examine developmental processes that generate well-defined and easily screenable morphological phenotypes. Conidiation plays a central role in the life cycle of many fungi and has been examined in greatest detail in Aspergitlus nidulans (Emericella niduIans) and N. crassa (Springer and Yanofsky 1989; Timberlake 1990; Springer 1993; Adams 1994). Other systems with special developmental features are also being investigated. In addition to producing conidia, Magnaporthe 9risea undergoes another form of asexual development to form specialized cells (appressoria) that are required for penetration of the host plant by this pathogen (Howard 1994).

2. Conidiophore formation in A. nidulans

Conidiation in growing colonies of A. nidulans is a programmed event that is relatively insensitive to nutrients and other environmental conditions, although light stimulates development (Timberlake 1990). Sensitivity to environmental conditions can be unmasked under certain growth conditions and in certain mutant backgrounds, For example, conidiation does not occur in submerged liquid culture unless induced by nutrient limitation or salt stress (Lee and Adams 1995). A. nidulcms only gains competence to initiate conidiation after 18 to 20 hours of growth as undifferentiated hyphae. Mutations that alter the time required to gain competence have been identified, indicating that the acquisition of competence is under genetic control.

The A. niduIans conidiophore is a highly ordered structure (Timberlake 1990) (figure 1). A specialized foot cell produces an aerial branch that grows to a height of approximately 100 ~em, whereupon the tip swells to form the conidiophore vesicle. Elongated metulae cells are formed by budding from multiple (roughly 60) sites on the vesicle surface. A tier of usually two phialide cells from each metula cell is produced.

Figure 1.

~ . Ce~

Phialide tula Vesicle

-~- Sl~lk

~ Foot Cell / __ f-Somatic Cell

. . . . . . . [ ) Conidiophore structure in AspergiIlus nidulans (Adams 1994).

M orphogenesis and vegetative differentiation 363

The nucleus of the phialide cell divides and the daughter nucleus moves toward the tip of the phialide. Inner cell wall material from the phialide cell is deposited around the nascent spore (conidium) as it pushes through the outer cell wall sheath of the phialide. The outer cell wall structure of conidia differs from the mother phialide cell. Additional conidia are budded from the phialide cell so that the oldest spores are at the distal tip of the proconidial chain.

2,1 Mutations affecting conidiation in A. nidutans

Fluffy (flu) and aconidial (aco) mutants do not initiate conidiation but do produce large masses of barren aerial hyphae, acoA, acoB and acoC and some of the f tu genes are required to achieve developmental competence. The f lu and aco mutants have pleiotropic defects and altered vegetative growth properties. The acoA, acoB and acoC mutants are also defective in sexual development (Tirnberlake 1990). A limited number of genetic loci have been found that block morphogenesis at defined stages during conidiation. The central regulatory pathway of conidiation (figure 2) is defined by three mutants, bristle (brIA) null mutants produce foot cells but aerial hyphae grow to 20 to 30 tirnes their normal length without forming vesicles, brlA is distinguished fiom f lu and aco mutants by the fact that there is no apparent effect of brIA mutation on vegetative mycelial growth, abacus (abaA) mutants produce conidiophore vesicles and initiate metulae cell differentiation, but they are defective in cell-type regulation and produce reiterations of phialide-like cells rather than true phialide cells and conidia. wet-white (wetA) rautants produce conidiophores; however, the conidia fail to mature and the defective cell walls eventually undergo lysis (Clutterbuck 1969; Timberlake 1990). Mutations specifically affecting spore and conidiophore colour and the hydrophobin rodlet layer are also known (Clutterbuck 1969; Adams 1994), but these auxiliary genes are not required for production of viable conidia.

Genes that modify conidial morphogenesis but are not part of the central regulatory pathway include stunted (stuA) and medusa (medA) (Clutterbuck 1969). stuA mutants

Developmental timeline

y

0h. 5 h. 10h 15hours Induction Vesicle Metulae and phialide Spore formation

formation formation and maturation

Class C and D 'Middle'

fluG ~ ~ wetA ftbA -E

Class A Class B 'early' 'Late'

Figure 2. Timeline of conidiophore formation and gene expression in Aspergillus nidulans.

364 Daniel J. Ebbole

produce short, thin-walled conidiophores and limited numbers of conidia, medA mutants have additional layers of metulae cells. These genes modulate expression of central pathway regulators (Adams 1994). stuA and medA mutants are self-sterile and so are also required for sexual development.

A homologue of the stuA gene, asm-1, has been cloned from N. crassa, asm-t mutants are female-sterile and have pleiotropic growth defects such as slow conidial germination and short (stunted) aerial hyphae that are capable of producing conidia (Aramayo et al. 1996). Thus there is some similarity in phenotype between stuA and asm-1 mutants, asm-1 and stuA have sequence similarity to S. cerevisiae PHDI and SOK2. These yeast genes are involved in regulating pseudohyphal growth.

2.2 Conidiation-specific genes of A. nidulans

The SpoC1 cluster represents a 38-kb region that codes for 20 transcripts. Eight transcripts accumulate in conidia and one accurnulates in phialide cells but not in conidia. The functions of the genes in the SpoC1, cluster are not known and deletion of the cluster does not cause a morphological defect. Chromatin structure might play a role in regulation of clustered conidiation-specific genes (Timberlake 1990). Targeted integration of the argB gene into any of several sites within the SpoC 1 cluster resulted in reduced basal expression of argB. In addition, movement of SpoC1 genes to ectopic sites resulted in their elevated expression in mycelia. However, ectopic integration did not prevent developmental induction of SpoC 1, suggesting that the genes in the cluster are subject to dual control A repressed chromatin state may prevent expression in mycelia and repression is relieved early in development. Developmental activation by the central regulatory pathway subsequently would activate expression to the appro- priate levels (Timberlake 1990).

A large set of conidiation-specific genes have been isolated. These genes have been grouped into classes A, B, C and D on the basis of the timing of their expression during conidiation (figure 2). They have also been classified according to which central regulatory genes are required for their expression (Adams 1994). brlA encodes a zinc- finger transcription factor and is required for full activation of the other central regulators. The brIA gene is subject to complex regulation at the transcriptional and translational levels (Adams 1994). abaA encodes another transcription factor that is part of a feedback loop that further activates brIA. brlA and abaA activate the early genes, wetA activates expression of the conidia-specific class B genes and its own expression. The combined activities of the three central regulators control expression of the class C and D genes that are important for phialide-specific functions.

In addition to activating conidiation-specific genes, expression of brlA was found to be sufficient to induce conidia formation in submerged hyphae. Although activation of abaA using an inducible promoter increased brlA expression, conidiation did not occur. Apparently activation of brtA directly is sufficient to initiate development but activation of brIA from its downstream regulator, abaA, is nonproductive.

2.3 Activation of the central pathway

fluG (also known as acol)) mutants are unable to conidiate on rich medium unless complemented extracellularly by a factor produced byfluG + strains (Lee and Adams

Morphogenesis and vegetative differentiation 365

1994a). fluG is therefore thought to be involved in production of a factor that stimulates conidiation. Consistent with this view, overexpression off luG in submerged cultures induces conidiophore development (Lee and Adams 1995).fIuG could be part of a system analogous to quorum sensing in bacteria where accumulation of homoserine lactone derivatives provides information about cell density to regulate gene expression (Swift er al. 1996). fluG mutants are able to conidiate on nutrient- limited medium. This suggests that fluG is part of a programmed developmental induction pathway, and when this pathway is defective alternative pathways for developmental induction are unmasked. Carbon starvation, but not nitrogen starvation, can induce conidiation in submerged cultures of the fIuG mutant (Lee and Adams 1995). Thus fluG may participate in activation of conidiation in response to a subset of inducing environmental signals such as nitrogen starvation in submerged cultures in addition to its role in programmed aerial development.

Additional loci that cause fluffy growth have been identified that do not properly induce brlA when exposed to air (Wieser e~ al. 1994). These are theflb (fluffy, low brlA expression) mutants, flbB, flbC, flbD and f lbE rnutants are delayed in conidiation but not affected in mycelial growth. The f lbA mutant is blocked in conidiation and the mutation also causes colonies to autolyse. The finding that overexpression offlbA (Lee and Adams 1994b) or flbD (Wieser and Adams 1995) activates brtA expression and induces development in submerged cultures indicates that these classes of genes play important roles in conidiation.

fIbA has sequence similarity to the S. cerevisiae SST2 gene (Lee and Adams 1994). SST2 regulates the activity of the G proteins involved in the pheromone response pathway. On the basis of this hint from yeast, it might be expected that G-protein-coupled receptors are involved in sensing signals that affect growth and development.

3. Macroconidiation in IV. c r a s s a

A. nidulans and N. crassa differ markedly in their manner of colony growth and conidial morphogenesis. While A. nidulans forms slowly spreading dense colonies on agar medium, N. crassa forms rapidly spreading (about ten-fold faster than A. ~idulans) and initially less dense colonies. Macroconidiation occurs rarely during the early colonization phase. After initial colonization is achieved (the edge of the Petri dish is reached) branching of the substrate mycelium accelerates and large numbers of aerial hyphae are formed that rnay then produce macroconidia. As in A. nidulans, macroconidiation in N. crassa occurs readily at an air interface and carbon or nitrogen starvation can lead to development in submerged culture (Plesofsky-Vig et at. 1983). Aerial macroconidiation is greatly influenced by environment and the number of macroconidiophores produced depends on carbon and nitrogen source (Ricci er al. 1991) and light (Ninnemann 1991). Macroconidiation is also under control of the circadian clock (Sargent and Kaltenborn 1972). Carbon dioxide inhibits macroconidiation, although the band (bd) mutant, often used in circadian-rhythm studies, is resistant to this effect (Sargent and Kaltenborn 1972). Growth on rich medium, such as yeast extract, represses macroconidiation. Even the manner of inoculation can modify colony morphology and conidiation pattern (Deutsch et al. 1993).

366 Daniel .1. Ebbole

Figure 3. Growing conidiophore of Neurospor~ crassa. Conidiophores branch fl'om aerial hyphae and initiate budding to form minor constrictions (m). Continued apical budding generates major constrictions (M). Apical budding (B) to form proconidial chains resembles yeast budding. (Photograph courtesy of K. Lee.)

There is no growth requirement for development of competence for macroconidiation. Germlings of any age can be harvested, and these develop aerial hyphae and conidia over a time course similar to that of harvested mycelium. Macroconidiation in N. crassa occurs on conidiophores that are unsophisticated compared to those of A. niduIans (figures 1 and 3) (Springer and Yanofsky 1989). A morphological switch from filamentous to budding growth occurs during apical extension of aerial hyphae. The process is similar to budding in S. cerevisiae, except that septation does not occur before the next round of budding begins. Frequently, branch formation in conidiophores is symmetrical (figure 3). During the initial transition to budding growth the septa that eventually form between cells have a diameter nearly equal to the diameter of the parent hypha. These are called minor-constriction chains and they are sometimes observed to revert to undifferentiated hyphal growth. Later, constrictions are pronounced and these are called major-constriction chains. Major-constriction chains are committed to budding growth and do not revert to minor-constriction- chain growth. Nuclei travel into the proconidial chains prior to septa formation and macroconidia are generally multinucleate.

3.1 The con genes

Several conidiation-specific genes have been characterized in N. crassa (figure 4). However, many con genes are regulated by factors in addition to macroconidiation. con-lO encodes an 86-amino-acid polypeptide that is induced at the time of major- constriction-chain formation. The CON10 protein has sequence similarity to the product of a starvation and stress-induced gene of Bacillus subtilis (Mueller et al. 1992) and likely accumulates in spores as a cellular protectant. CON10 accumulates in macroconidia and microconidia, and ascospores (Springer 1993). In the mycdium, con-lO is also regulated by light and the circadian clock independently of developmental control (Lauter and Yanofsky 1993). con-IO expression is repressed in mycelia and activated during development (Corrochano et aI. 1995), like the SpoC1 duster in A. nidulans.

M orphogenesis and vegetative differentiation

Developmental timeline

Oh 4 h

Induction minor chains

con-8 ea$

con-6

8h

rrlajor chains

con- 10 con- 13

1 2_ h 1 6 hours

septa conidial formation separation

fl gran csp- 1 acon-3 tng csp-2

Timeline of conidiophore formation and gene expression in Neurospora crassa.

fld acon-2

Figure 4.

367

3.2 'Classical' macroconidiation mutants

Many morphological mutants of N. crassa have been isolated that simultaneously affect colony morphology, hyphal growth characteristics and development (Perkins et al. 1982). Relatively few nmtants have been isolated that specifically affect macroconidiation and con gene expression (figure 4). aconidate-2 (acon-2) andfhtffyoid (fld) mutants do not produce minor constriction chains.fluffy (f/) and acon-3 mutants form minor but not major constriction chains, f l mutants are widely used by Neurospora geneticists because f l female parents have enhanced fertility, conidial separation-1 (csp-t) and csp-2 mutants form normal comdiophores, but these do not mature to release free conidia. Other mutants are known that alter budding pattern. Typically, one to two buds emerge from the growing tip cell of the proconidial chain, but cjran mutants produce multiply budded cells. The tangerine (tug) mutant is unable to arrest growth or initiate new budding in a fraction of proconidia and can produce gigantic macroconidia (Springer and Yanofsky 1989).

3.3 Macroconidiation mutants with enhanced con gene expression, rco-1

Regulators of con gene expression and macroconidial morphogenesis have been obtained by selecting for mutants that express the con-lO and con-6 genes during vegetative growth (Madi et aI. 1994). A number of mutants were obtained that were defective in conidiation. Why activation of conidiation-specific gene expression should result in a block in conidiation is uncertain. However, it is possible that premature actiwttion of genes that are normally expressed late during conidiation may block early functions required for proper development.

rco-i mutations are pleiotropic and alter vegetative growth, macroconidiation and sexual development (Yamashiro et al. 1996). Null mutants are blocked in macroconidiation at the conidial separation stage, like csp mutants. The rco-1 ~ allele displays a more severe conidiation phenotype and is blocked prior to minor-constriction-chain formation. The rco-1 gene has sequence similarity to the yeast TUP1 gene. TUP1 forms part of a transcriptional repression complex that regulates many classes of genes in S. cerevisiae, including genes regulated by cell type (Mukai et aI. 1991). rco-1 may act directly or indirectly through a mycelial repression site identified in the con-lO promoter


(Corrochano et al. 1995). In S. cerevisiae, the TUP1 repression complex has been shown to alter chromatin structure in the promoter regions of regulated genes (Edmondson et aI. 1996). Thus mycelial repression of con-lO expression and the A. nidulans SpoC1 cluster may be brought about by similar mechanisms involving chromatin structure. Interestingly, con-lO is tightly linked to several other con genes and is adjacent to con-13, a conidiation- specific gene that is expressed during development at the same time as con-lO.

3.4 Microconidiation in N. crassa

Microconidia are usually hidden by the massive canopy of macroconidiophores produced under most culture conditions. Microconidiation is usually examined in f l mutants that have additional modifying mutations that enhance microconidiation. However, microconidiation is readily observed in wild type by washing macroconidia from the surface of a 10-day-old Petri plate culture on nitrate minimal medium. Microconidiophores develop over the course of one to two days and no new macroconidiation is observed. Specialized aerial hyphae are produced from a 'foot cell' in the substrate mycelium. These give rise to highly branched microconidiophores, each capable of producing thousands of microconidia (figure 5). The branched aerial hyphae

Figure 5. Microconidiophore formation in wild-type Neurospora crassa. A. The foot cell (F) forms an aerial branch with a diameter greater than that of substrate hypha. Septa in microconidiophore branches are approximately 10/~m apart and each phialide cell produces microconidia (C) from a single site. B. Older microconidiophore produces many microconidia (C) and a collarette of outer cell wall material is visible where the microconidia erupt through the outer cell wall (E). C. View of entire mature aerial microconidiophore showing highly branched structure. Microconidia (C) are small (1-4 #m) compared to macroconidia (5-10 #m) (M). D. View of microconidiophores (P) oil agat surface stained with cotton blue dye.

M orphogenesis and vegetative differentiation 369

are partitioned into phialide cells by septa that are 10 #m apart. The outer wall of the microconidium is produced from the inner cell wall material of the phialide cell in much the same manner as conidia of A. nidulans (Springer 1993). Microconidia are uninuc- leate and have poor viability--typically 5 to 30% of the microconidia produced are capable of germinating.

No detailed genetic or molecular analyses of microconidiation have been performed other than identification of mutations that enhance microconidiation. The microcycle microconidiation (mcm) locus is of particular interest since it controls microconidiation in a conditional manner (Maheshwari 1991). Many of the con genes expressed during macroconidiation are also expressed in microconidia (Springer 1993). It is not known whether the same regulators control con gene expression during macroconidiation and microconidiation.

4. Conidiation in M. grisea

Genetic and molecular techniques used for N. crassa and A. nidulans can be applied to M. grisea, making it an excellent model system for the study of a plant pathogen. Conidiation is strongly influenced by environmental conditions such as nutrients and light (Barksdale and Asai 1961; Shi and Leung 1995). Aerial hyphae produce a short stalk from which the proconidium is produced by budding of a single cell. As the primary budded cell matures, septa are laid down to produce a tear-drop- shaped conidium with three cells (figure 6). Three to seven conidia are typically produced by a single aerial conidiophore. The entire length of the conidium is roughly 20 pm, with the fat end of the spore (about 8 #m wide) attached to the stalk. A compartment at the tip of the conidium contains an adhesive, termed spore-tip mucilage, that is released upon hydration to attach the conidium to solid substrates (Howard 1994).

Mutants defective in production of conidia have been isolated, but most have pleiotropic defects. Several of the con (conidiation) mutants have been characterized and have defects in aerial growth, conidia formation and conidia morphology (Shi and Leung 1995). Some of the mutants are altered in growth rate, fertility, appressorium

Figure 6. Conidia(C)ofMagnaporthegriseagerminatetoformappressoria(A).fPhotograph courtesy of J. L. Beckerman.)

370 Daniel J. EbboIe

formation and response to light. Additional spore morphology (Smo-) mutants arise spontaneously at high frequency and affect the shape of conidia, appressoria and asci, but do not affect ascospore shape (Hamer etaI. 1989). Hence, as in A. nidulans and N. crassa, there may be many morphological modifiers with pleiotropic effects in M. grisea but relatively few mutations that block defined stages in development. Determining whether regulators of conidiation in N. crassa or A. nidulans have homologues in M. 9risea is of importance and might facilitate identification of key genes.

4.1 Formation of the M. grisea appressorium

M. grisea produces a specialized cell used to penetrate the surface of its host plant. Germination usually occurs from either or both of the terminal cells of the conidium. Approximately 6 to 8 hours following deposition on an artificial surface, appressoria (figure 6) are produced. The appressoriurn has several cell wall layers including a layer composed of melanin. The melanin layer forms a barrier through which water, but not solutes, can pass. An increase in solute concentration due to the breakdown of glycogen causes water to enter the cell by osmosis. The pressure increase is used to help force a hypha into the host cell (Howard 1994).

Appressoria only form when in contact with plants or certain artificial substrates. The precise surface requirements for signalling appressorimn formation are not fully understood. Hydrophobicity has been implicated as a critical surface para- meter and essentially all hydrophobic surfaces are inductive (Lee and Dean 1994). However, some hydrophilic surfaces are also inductive. Certain chemicals that may mimic plant wax or cutin monomers, such as 1, 16-hexadecanediol, induce appressorium formation in M. grisea on noninductive surfaces (Gilbert et al. 1996). Hence there may be alternative routes to signalling appressorium formation. cAMP also induces appressorium formation on noninductive surfaces and this suggests that a cAMP-dependent signalling pathway regulates appressorium formation (Dean eral. 1994). cAMP-dependent protein kinase rnutants of M. grisea exhibit delayed formation of nonfunctional appressoria (Xu et al. 1997; Mitchell and Dean 1995). Competence to form appressoria in response to an inductive surface occurs during a narrow period of time (Beckerman and Ebbole 1996). If appressorium formation is repressed during the first eight hours of growth, it does not occur at a later time, even in the presence of cAMP.

There are several mutants known that are specifically blocked in appressorium formation (Dean etal. 1994). Appressorium formation in some of these mutants can be rescued by cAMP, suggesting that they possess defects in sensing inductive cues. Cloning of signal transduction components by PCR followed by gene disruption has identified a MAP kinase specifically required for appressorium formation (Xu and Hamer 1996). This suggests that a cAMP-dependent pathway and a MAP kinase cascade both contribute to appressorium development.

A limited number of genes expressed during appressofium formation have been cloned. One pair of genes was identified by differential hybridization (Dean et al. 1994). A gone encoding a fungal hydrophobin, MPGI, was identified independently as agene expressed during both conidiation and pathogenesis. It was found that MPGI plays a role in appressorium formation (Talbot et al. 1996).

Morphogenesis and vegetative differentiatio~ 371

5. Fungal hydrophobins in morphogenesis

Hydrophobins are important determinants of morphogenesis in filamentous fungi: as structural components of the cell wall and by virtue of their role in environment sensing. Hydrophobins are small, cysteine-rich and generally hydrophobic polypept- ides. Under inducing conditions they are secreted in copious amounts. Hydrophobins are able to self-assemble into rodlet structures at hydrophobic-hydrophilic interfaces (Wessels 1996). Rodlets can be found to decorate conidia and aerial hyphae where they alter the hydrophobic characteristics of the cell surface. Aerial hyphae and conidia coated with rodlets are difficult to wet but hydrophobin mutants are easily wettable. In N. crassa and A. nidulans the hydrophobin rodlet layer aids in aerial dispersal of conidia. In Schizophyllum commune hydrophobins are critical for aerial growth from stationary liquid cultures (Wessels 1996). Thus hydrophobins could be classified as morphogens whose accumulation induces aerial development. Hydrophobins may also be involved in cell-cell adhesion during development of the fluiting body. In addition hydrophobins coat the surface of hyphae that line air spaces in the S. commune fruiting body and this hydrophobic coating may be critical in allowing gas exchange by preventing water soaking of the air spaces (Wessels 1996).

Hydrophobins can act as morphogenetic determinants of development in an aque- ous environment. Under certain conditions appressorium formation in M. grisea is dependent on expression of the MPG1 hydrophobin. In this case it is thought that the assembly of hydrophobin rodlets at the interface of the germ tube with the hydrophobic plant surface can signal appressorium formation. The mechanism for this signalling may be increased adhesion of the hypha to the substrate, mediated by the rodlet layer (Talbot et al. 1996). It is speculated that this adhesion might activate mechanosensitive ion channels to influence cAMP levels (Dean et al. 1994; Wessels 1996).

6. Conclusion

Signalling of asexual development can be a prograrnmed event as observed in A. nidulans, wherefhtG may play an important role in generating a signal to induce conidiation. However, nutritional status and light are important environmental signals that influence conidiation. Nutritional regulation of conidiation is observed in A. niduIans, but it is normally masked by the developmental program that is superim- posed on environmental control. When the developmental program is inactive (by certain mutations or growth in submerged culture), nutritional control mechanisms can function. In M. grisea appressorium formation, development is programmed in that it can be induced only during a narrow time window of developmental competence. However, the program must be activated by an environmental stimulus, such as substrate characteristics. Macroconidiation in N. crassa and conidiation in M. grisea are highly responsive to the environment. Learning how fungi sense environmental and endogenous signals for cellular differentiation is a major area of current exploration.

Genetic studies of conidiation are leading to new insights concerning signN transduction pathways involved in development. Continued analysis of fluffy mutants and their suppressors in A. nidulans will provide an unparallelled genetic understanding of the events controlling conidiation in a filamentous fungus. Comparison of the functions of homologous genes between organisms is needed to help define the common and


unique features of development for individual fungal species. The cloning of the stuA homologue, asm-I, is an initial step in this direction. The identification of a MAP kinase that specifically blocks appressorium formation demonstrates that general analysis of components of signal transduction pathways can be a useful approach as well.

Clustering of development-specific genes is seen in both A. nidulans and N. crassa. Repression of clustered genes through an inactive state of chromatin is possibly a common mechanism for control. Activation of development-specific genes occurs by control of the timing and localization of transcription factor activity, as observed in conidiation in A. nidulans. Additional studies of the promoters of developmentally regulated genes and the trans-acting factors that control transcription are needed.

Many examples have been presented in which a single mutation affects multiple developmental pathways. Thus, as in S. cerevisiae, there are shared components for different developmental processes. In any one organism it is necessary to understand how specific signalling pathways are activated and how signalling pathways interact. Identifying the conserved mechanisms used by fungi in signalling alterations in growth polarity, hyphal branching and orientation of hyphal tip growth is an important challenge. Defining how fungal species differ in their orchestration of these conserved signalling mechanisms will explain their different morphologies.

References

Adams T. H. 1994 Asexual sporulation in higlner fnngi. In The growing fungus (eds.) N.A.R. Gow and G. M. Gadd (London: Chapman and Hall) pp. 367 382

Alex L. A., Borkovich K. A. and Simon M. I. 1996 Hyphal development in Neurospora crassa: Involvement of a two-component histidine kinase. Prec. Natl. Acad. Sci. USA 93:3416--3421

Aramayo R., Peleg Y., Addison R. and Metzenberg R. 1996 Asm-I +o a Neurospora crassa gene related to transcriptional regulators of fungaI development. Genetics 144:991-1003

Barksdale T. H. and Asai G. N, 1961 Diurnal spore release of PMcularia oryzae from rice leaves. Phytopathology 51 : 313-317

Beckerman J. L. and Ebbole D. J. 1996 MPG 1, a gone encoding a fungal hydrophobin of Magnaporthe grisea, is involved in surface recognition. Mol. Plant-Microbe Interact. 9:450-456

Bruno K. S., Aramayo R., Minke P. F.0 Metzenberg R. L. and Plamann M. 1996 Loss of growth polarity and mislocalization of septa in a Neurospora mutant altered in the regulatory subunit of cAMP-dependent protein kinase. EMBO J. 15:5772-5782

Chen B., Gao S., Choi G. H. and Nuss D. L. 1996 Extensive alteration of fungal gene transcript accumtdation and elevation of G-protein-regulated cAMP levels by a virulence-attenuating hypovirus. Proc. Natl, Acad. Sci. USA 93:7996-8000

Cid V. J. 1995 Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. MicrobioJ. Rev. 59:345-386

Clutterbuck A. J. 1969 A mutational analysis of conidial development in Aspergillus nidulans. Genetics 63: 317 -327

Corrochano L. M., Lauter F.-R., Ebbole D. J. and Yanofsky C. 1995 Light and developmental regulation of the gone con-lO of Ne~rospora crassa. Dev. Biol. 167:190-200

Dean R. A., Lee Y.-H., Mitchell T. K. and Whitehead D. S. 1994 Signalling systems and gone expression regulating appressorium formation in Magnaporthe grisea. In Rice blast disease (eds.) R. S. Zeigler et al. (Wallingford: CAB International) pp. 23-34

Deutsch A., Dress A. and Rensing L. 1993 Formation of morphological differentiation patterns in the ascomycete Neurospora crassa. Mech. Dev. 44:17-31

Edmondson D. G., Smith M. M. and Roth S. Y. 1996 Repression domain of the yeast global represser Tupl interacts directly with histories H3 and H4. Genes Dev. 10:1247-1259

Gilbert R. D., Johnson A. M. and Dean R. A. 1996 Chemical signals responsible for appressorium formation in the rice blast fungus Maqnaporthe grisea. Physiol. Mol. Plant Pathgl. 48:335-346

M o r p h o g e n e s i s and vegetat ive dif-ferentiation 373

Hamer, J. E., Valent B. and Chumley F. G. 1989 Mutations at the SMO genetic locus affect the shape of diverse cell types in the rice blast fungus. Genetics 122:351-362

Herskowitz I. 1995 MAP kinase pathways in yeast: for mating and more. Celt 80:187-197 Howard R. J. 1994 Cell biology of pathogenesis. In Rice blast disclose (eds.) R. S. Zeigler el el. (Wallingford:

CAB International) pp. 3-22 Lauter F.-R. and Yanofsky C. 1993 Day/night and circadian rhythm control of con gone expression in

Ncurospora. Proc. Natl. Acad. Sci. USA 90:8249-8253 Lee B. N. and Adams T. H. 1994a The Aspergiltus ~idulans.fluG gone is required for production of an

extracellular developmental signal and is related to prokaryotic glutamine synthetase I. Genes Dev. 8: 641-651

Lee B. N. and Adams T. H. 1994b Overexpression of f lbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and l?remature initiation of development. Mol. Microbiol. 14: 323~-334

Lee B. N. and Adams T. H. 1995 .]'lug and f IbA function interdependently to initiate conidlophore development in Asperyillus nidulans through brlA[:J activation. EMBO J. 15:299-309

Lee Y.-H. and Dean R. A. 1994 Hydrophobicity of contact surface induces appressorium formation in Magnaporthe ~,lrisea, FEMS Microbiol. Lett. 115:71-76

Madi L., Ebbole D. J., White B. T. and Yanofsky C. 1994 Mutants of Neurospora crassa that alter gone expression and conidia development. Proc. Natl. Acad. Sci. USA 91:6226-6230

Maheshwari R. 1991 Microcycle conidiation and its genetic basis in New'ospora crctssa. J. Gem Microbiol. 137:2103 2115

Mitchell T. K. and Dean R. A. 1995 The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Maonaporthe grisea. Plant Cell 7: 1869-1978

Mueller J.P., Bukusoglu G. and Sonenshein A, L. 1992 Transcriptional regulation of Bacillus subtilis glucose storvatien-inducible genes: control of ~,jsiA by the eomP-comA signal transduction system. J. Baeteriol. 174:4361-4373

Mukai Y., Harashima S. and Oshima Y. I991 AARI/TUP1 protein, with a structure similar to that of the beta subunit of G proteins, is required for al-alpha 2 and alpha 2 repression in cell type control of Saccharomyces cerevisiae. Mol. Cell. Biol, 1 l: 3773-3779

Ninnemann H. 1991 Photostimulation of conidiation in mutants o1" Neurospora erassa. J. Photochem. Phozobiol. B: Biol. 9:189-199

Perkins D. D., Radford A., Newmeyer D. and Bjorkman M. 1982 Chromosomal loci of Neurospora crassa. Microbiol. Roy. 46: 426-570

Plamann M., Minke P, F., Tinsley J. H. and Bruno K. S. 1994 Cytoplasmic dynein and actin-related protein Arpl are required for normal nuclear distribution in filamentous fungi. J. Cell Biol. 127:139-149

Plesofsky-Vig N., Light D. and Brambl R. 1983 Paedogenetic conidiation in Neurospora erassa, Exp. Mycol. 7:283-286

Ricci M., Krappmann D. and Russo V. E. A. 1991 Nitrogen and carbon starvation regulate conidia and protoperithecia formation of Neurospora crassa. Fungal Genet. Newsl, 38:87-88

Sargent M. L, and Kaltenborn S. H. 1972 Effects of medium composition and carbon dioxide on circadian eonidiation in Neurospora. Plant Physiol. 50:171-175

Shi Z. and Leung H. 1995 Genetic analysis of sporulation in Magnaporthe arisea by chemical and insertional mutagenesis. Mol. Plcmt-Microbe Interact. 6:949-959

Springer M. L. 1993 Genetic control of Fungal differentiation: The three sporulation pathways of Ne~Lraspora crassa. BioEssays 15:365-374

Springer M. L. and Yanofsky C. 1989 A morphological and genetic analysis of conidiopl~ore development in Neurospora erassa. Genes Dev. 3:559-571

Swift S,, Throup J. P., Williams P., Salmond G. P. C. and Stewart G. S, A. B. 1996 Quorum sensing: a population-density component in the determination of bacterial phenotype. Trends Biochem. Sci. 21: 214-219

Talbot N. J., Kershaw M. J., Wakley G. E., de Vales O. M. H., Wessels J. G, H. and Haraer J, E. 1996 MPG1 encodes a fungal hydrophobin involved in surface interactions during infection-related development of Magnaporthe grisea. Plant Cell 8:985-999

Timberlake 1990 Molecular genetics of Aspcrgiilus development. Am~u. Roy. Genez. 24:5-36 Turner G. E. and Borkovich K. A. 1993 Identification of a G Protein g subunit from Neurospora crassa that is

a member of the G~ family. J. Biol. Chem. 268:14805-14811


Wessels J. G. H. 1996 Hydrophobins: Proteins that change the nature of the fungal surface. Ad~. Microb. Physiol. 38:1-45

Wieser J. and Adams T. H. 1995 flbD encodes a myb-like DNA-binding protein that coordinates initiation of Aspergilh~s nidulal~s conidiophore development. Genes Dev. 9:491-502

Wieser J., Lee B. N., Fondon J. W. III and Adams T. H. 1994 Genetic requirements for initiating asexual development in Aspergillus nid~tans. Curr. Genet. 27:62-69

Xu J.-R. and Hamer J. E. 1996 MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Maqnaporthe 9risea. Genes Dev. 10:2696-2706

Xu J.-R., Urban M., Sweigard J. A. and Hamer J. E. 1997 the CPKA gone of Magnaporthe 9risea is essential for appressorial penetration. MoI. Plant-Mic~'obe Intepact. 10:187-194

Yamashiro C.T., Ebbole D. J., Lee B.-U., Brown R. E., Bourland C., Madi L. and Yanofsky C. 1996 Characterization of reo-1 of Neurospora crassa, a pleiotropic gone affecting growth and development that encodes a homologue of Tupl of Saccharomyces cerevisiae. Mol. Cell. Biol. 16:6218-6228

Yarden O., Plamann M., Ebbole D. J. and Yanofsky C. 1992 co~-1, a gene required for hyphal elongation in Neurospora cpassa, encodes ~ protein kinase. EMBO J. 11:2159-2166

Documents

Morphogenesis and vegetative differentiation in