Phenotypic plasticity in fungi: a review with observations onAureobasidium pullulans

Ralph A. SlepeckyWilliam T. Starmer1

Department of Biology, Syracuse University, Syracuse,New York 13244

Abstract: Phenotypic plasticity in fungi is reviewed inthe context of observations on phenotypic changes inthe colony morphology of the fungus Aureobasidiumpullulans. The variation in colony form is shown todepend on (i) the types of single carbon substrates(sugars and sugar alcohols) used in the growthmedium, (ii) colony age, (iii) incubation tempera-ture, (iv) light cycle and (v) substrate type. Expandingcolonies grow in a developmental sequence that showsynchronize growth phase shifts as well as unusualtransitions from homogeneous to sectored, yeast tomycelial and giant to microcolonial growth forms.Epigenetic influences on phenotypic switches aresuggested to be potential causes of form changes. Thedesirable properties of a model organism for studyingphenotypic plasticity are discussed and past work onthe yeast-mycelial transition of fungi is reviewed.

Key words: epigenetics, model organism, yeast-mycelial transition

INTRODUCTION

Fungi are notable for their ability to switch growthforms in response to environmental stimuli (Raynerand Coates 1987). Most likely fungi rely on thecapacity to make these shifts to achieve survival,dispersal and reproductive advantages, and no doubttheir success at these fundamental processes helpsexplain their recognition as a kingdom. The ability offungi to alter forms and shift to different modes ofliving has been of interest to mycologists because oftheir importance to understanding fungal molecularbiology, ecology and evolution as well as their utilityin industry and their role in both infection andbiological control. One fungus that assumes manydifferent shapes (i.e. it is pleomorphic) and lives in awide variety of habitats is Aureobasidium pullulans.This fungus has been recovered from diverse surfacestypes, especially the phylloplane (Andrews et al 2002,McGrath and Andrews 2007, Andrews and Harris1997, Woody et al 2007). Examples of other surface

sources include glass (Schabereiter-Gurtner et al2001), painted material (Shirakawa et al 2002), aswell as rocks and marble (Urzı et al 1999, 2001). It isfound in soil, freshwater and saltwater, ice (Zalar et al2008) and is commonly recovered from the atmo-sphere (e.g. Shelton et al 2002, Lugauskas et al 2003,Griffin et al 2003, Samson et al 2004) and above (i.e.the Mir space station, Alekhova et al 2005). Unusualsources of A. pullulans, often as a contaminant,include for example samples containing ancient DNA(Hauf et al 1995), aviation fuel (Rauch et al 2006),spacecraft (La Duc et al 2003) and damaged nuclearreactors (Zhdanova et al 2000). Aureobasidium pull-ulans is involved as the principal colonizer initiatingbiodeterioration (e.g. plasticized polyvinyl chloride,Webb et al 2000) has been used as an indicator ofenvironmental pollution (Deshpande et al 1992) andis implicated in human disease (Taylor et al 2005).

The pleomorphic characteristic of fungi (Savile1969) is also know as ‘‘phenotypic plasticity’’, that isthe ability of any organism to respond to environ-mental signals by altering morphology, physiologicalstate or behavior (West-Eberhard 1989). This ability iswidespread among taxa and has been studiedextensively primarily because of its importance to anorganism’s ability to survive and propagate. Thefunction that describes the range of phenotypesproduced by a single genotype in a suite ofenvironments is called a ‘‘reaction norm’’ and is aconcept generally adopted by geneticists studyingevolution and ecology (Pigliucci 1996). Because overtheir lifetimes organisms occur in changing environ-mental conditions they are expected to have reactionnorms that scale to the variable environment theyinhabit and thus the individual is expected to bephenotypically plastic. What determines the shape ofthe reaction norm and how the change from onephenotype to another occurs are central questions inmolecular, evolutionary and ecological genetics.

Pigliucci (1996) discusses two broad approaches tostudying phenotypic plasticity. One is statistical, whichuses the tools developed by students of quantitativegenetics. The major limitation of this method is thatthe assumptions underlying the theory are often toosimple and as a consequence inferences about geneticmechanisms can be unrealistic. The second approachis a mechanistic study of the genes involved inphenotypic plasticity. The initial phase of thisapproach is to use a genetic screen designed to

Accepted for publication 7 May 2009.1 Corresponding author. E-mail: wstarmer@syr.edu

Mycologia, 101(6), 2009, pp. 823–832. DOI: 10.3852/08-197# 2009 by The Mycological Society of America, Lawrence, KS 66044-8897

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detect plasticity genes or genetic networks involved inthe transition from one phenotype to another. Tofacilitate this type of work model plants and animals,such as Arabidopsis thaliana or Drosophila melanoga-ster, often are employed as experimental organisms.

Even though microorganisms have been used forstudies in plasticity (e.g. Promislow 2005, Stomp et al2008), their great potential for understanding themechanism of phenotypic plasticity have not beengenerally recognized, especially in fungi ( Jennings1993, Andrews 1992, Bago et al 2004). Bacterialcolonies growing on the surfaces in Petri dishes showdifferentiated structures that result from a complexseries of morphological events. The geometry ofbacterial colonies can be a consequence of swarming,chemotactic auto-aggregation, self-engineering, inter-cellular communication, nutrient gradients and stress(Shapiro 1995, Ben-Jacob and Levine 2006). Shapiro(1998) emphasized the need to consider a bacterialpopulation as a multicellular organism with complexsignaling systems that result in coordinated behaviors.These emergent phenotypes of single cells growingtogether and communicating affect survival, move-ment and reproduction, all of which can be beneficialand thus evolve. Pattern formation in bacteria, such asBacillus subtilis (Mimura et al 2000), exemplify thedegree of phenotypic plasticity that can occur inmicroorganisms grown in relatively simple cultureconditions. In this case colony morphology showcharacteristics of ‘‘phase transitions’’ where there areabrupt changes from one morphologic type toanother along nutrient and agar density gradients.

We report here that the fungus Aureobasidiumpullulans reversibly forms different types of coloniesdepending on the substrate and temperature onwhich it is grown, that is its environment. Thisproperty coupled with other natural attributes sug-gests that this microorganism could serve as a modelfor investigating a diversity of problems on the causesof phenotypic plasticity. Along with our observationson A. pullulans we discuss and review (i) the desirableproperties of a model organism, (ii) dimorphism infungi, (iii) responses to light and (iv) epigeneticinfluences on phenotypic plasticity.

MATERIALS AND METHODS

The source of inocula was a single strain of A. pullulans (04-313.1) isolated from beetles in morning glory flowers,Massai Kopjes, Serengeti National Park, in 2004. Most of thegrowth media were single-carbon sources in agar. A detaileddescription of these media is given by Yarrow (1998). Inessence growth tests for carbon assimilation were conductedon medium in 95 mm glass Petri dishes composed of 2%

Bacto agar, 0.67% YNB (Bacto yeast-nitrogen base) and0.5% or 2.0% (w/v) carbon source. The initial inoculum

(,104 yeast-phase cells suspended in 1 mL H2O) was takenfrom a culture growing on YNB glucose (0.5%) agar and wasplaced on the center of the agar medium in the Petri dish.The inoculated plates were sealed around the rims withparafilm. Colonies were grown 2–3 d up to 3–4 mo at 9, 20or 25 C. Photographs were taken under standard conditionsat various intervals over the period. Additional growth testswere conducted under (i) constant light or dark and alight : dark (12 h : 12 h) photoperiod, (ii) with inocula fromcolonies growing on different sugar sources, (iii) usingdifferent nitrogen sources added to YCB (Bacto yeast-carbon base) and (iv) conditions that potentially demon-strate epigenetic influences on changing colony morphol-ogy. Experiments in this latter category were conducted bysupplementing with 20 mM nicotinamide or 20 mM nico-tinic acid in buffered (100 mM phosphate) medium.Nicotinamide (the amide of nicotinic acid) is known toinhibit histone deacetylases that are essential for epigeneticregulation of gene expression (Prusty et al 2008). Howevermedium supplemented with nicotinic acid is not expectedto produce epigenetic changes, and thus morphologicalshifts that occur on medium with nicotinamide but not onmedium containing nicotinic acid can be a useful indicatorof epigenetic control. The major strain in this study wassubjected to PCR amplification and rDNA sequencing asdescribed by Lachance et al (2005).

RESULTS

We provided examples of the diversity of morpholog-ical (colony) responses. Colony morphology is rela-tively uniform after 4 wk on 0.5% glucose at 25 C(FIG. 1A). Growth at 9 C (FIG. 1C, D) producesconsiderable sectoring (i.e. sectors with melanin)and distinct stages of growth if allowed to grow 12 wk.Although relatively minor there was a distinct changein response to light (FIG. 1B) with daily growth ringsapparent as the colony expanded for 3 wk. This photoresponse is immediately interrupted if the colony isshielded from light and allowed to grow in completedark in the same incubator. We recorded the effect ofincreasing the concentration of glucose 0.5–2%

(FIG. 1E). In this case there are manifest changes inthe growth pattern with discrete dynamic phases ofgrowth as the colony expands. We recorded the effectof growth on 2% glucose supplemented with 20 mMnicotinamide (FIG. 1F). The colony under theseconditions is more similar to the growth on the lowerconcentration (i.e. 0.5% glucose, FIG. 1A) and mightindicate an epigenetic effect.

Colony morphology was a spreading black (mela-nin) branching structure after 4 wk on 0.5% dulcitolat 25 C (FIG. 2A). Influences of temperature and timewere recorded (FIG. 2F, G). Growth at 9 C wasrecorded (FIG. 2F, G), which indicated that little tono melanin accumulates at low temperature after4 wk and somewhat more melanin is present after

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12 wk. We also recorded growth on 0.5% (FIG. 2B, C)and 2% (Fig. 2D, E) dulcitol respectively. Coloniesgrown on media supplemented with 20 mM nicotin-amide were recorded (FIGS. 2B, D), as were thosegrown in media supplemented with 20 mM nicotinicacid (FIGS. 2C, E). In this case an epigenetic effect isimplicated because the amide is expected to inducechanges in the chromatin while the acid is not andshould appear like the colony (FIG. 2A) on mediumthat was not supplemented. Note that this is true foreither concentration of dulcitol.

Colony morphology was noted (FIG. 3) on othercarbon sources (0.5%, A 5 salicin, B 5 glycerol and C5 i-erythritol) when incubated 4 wks at 25 C (column1) and 4 wk at 9 C (column 2) or 12 wk at 9 C(column 3). Lower temperature resulted in moremelanin and in some cases (glycerol and i-erythritol)dynamic developmental phases of growth. Theseexamples illustrate the diverse and complex changes

that occur over time and as a consequence ofalterations in simple carbon sources.

The colonies (FIGS. 1, 2, 3) were grown from asuspension of cells from a single strain of A. pullulansgrown on YNB + 0.5% glucose agar. Experimentsconducted with inocula from colonies grown on fourdifferent carbon sources (0.5% w/v, glucose, dulcitol,mannitol and salicin) each transferred to all fouroriginal sources showed that morphological changesexperienced on the original plates were reversible.Not all tests are shown, but other carbon and nitrogensources showed diverse phenotypes and changes overtime. Tests with other strains of A. pullulansproduced similar results. This was expected andindicated that intraspecific variation in phenotypicplasticity also exists. Partial rDNA sequence of thestrain (04-313.1, GenBank: FJ744598) in these studieshad strong similarity (greater than 98% similarity) tomany sequences from different strains of A. pullulans

FIG. 1. Colonies of A. pullulans cultured on YNB +glucose. A 5 4 wk at 25 C, 0.5% glucose; B 5 3 wk at 20 C,0.5% glucose, 12 h light : 12 h dark photoperiod (back andfront illuminated). Colonies grown under continuous lightor dark on the same medium appear similar to the colonyin A. C and D 5 0.5% glucose at 9 C for 4 and 12 wkrespectively; E 5 2.0% glucose at 25 C, F 5 2.0% glucose at25 C supplemented with 20 mM nicotinamide. Each photois 5 cm square; bar: A 5 1 cm.

FIG. 2. Colonies of A. pullulans cultured on YNB +dulcitol. A 5 4 wk at 25 C, 0.5% dulcitol; B and C 5 4 wk at25 C, 2.0% glucose, supplemented with 20 mM nicotin-amide and 20 mM nicotinic acid respectively. Coloniesgrown at 25 C without supplements appear similar to thecolony in A. D and E 5 4 wk at 25 C, 0.5% glucose,supplemented with 20 mM nicotinamide and 20 mMnicotinic acid respectively. Colonies grown at 25 C withoutsupplements appear similar to the colony in A. F and G 5

0.5% glucose at 9 C for 4 and 12 wk respectively. Each photois 5 cm square; bar: A 5 1 cm.

SLEPECKY AND STARMER: PHENOTYPIC PLASTICITY IN FUNGI 825

deposited in GenBank. The strongest similarity (99%)was with group 3 and 4 strains defined respectively asA. pullulans var. subglaciale and var. namibiae by Zalaret al (2008).

DISCUSSION

Colony changes due to mutagenesis were importantin early studies in microbial genetics. For example thefact that DNA or its modification is the geneticdeterminant was revealed by colony changes inpneumococcus (Avery et al 1944). However pheno-typic change (not due to treatment with mutagens) incolonies is not often observed, possibly because purecultures have only a single characteristic colonymorphology or variation in colony type is seen onlyamong strains. However fungi are well known fortheir extreme phenotypic plasticity (Slutsky et al 1985,Andrews 1992, Bago et al 2004) and have propertiesthat make them suitable for laboratory studies of howand why phenotypes change when environmentalconditions change. It is widely thought that fungidevelop differentially to explore and exploit theenvironment and to respond quickly by changingmycelia when environmental conditions change. Forexample the nematode-trapping fungus Arthrobotrysoligosopora develops different structures from myceliafor infecting and parasitizing nematodes or otherfungi. Their response to environmental signals canresult in structures for catching nematodes, such as

adhesive nets and conidial traps, and for mycoparasit-ism by developing hyhal coils and appressoria(Nordbring-Hertz 2004). By far the most extensivelystudied phenotypic change in fungi is the yeast-mycelial (YM) transition. Several reviews of dimor-phism in fungi discuss general features (Scherr andWeaver 1953, Romano 1966, Bemmann 1981), phasetransitions and cell differentiation (Maresca andKobayashi 1989), signaling (Lengeler et al 2000, Leeet al 2003), quorum sensing (Nickerson et al 2006),regulation (Rayner and Coates 1987) and pathoge-nicity in plants and animals (Rippon 1980, Madhaniand Fink 1998, Lee et al 2003, Nadal et al 2008,Morrow and Fraser 2009).

Desirable properties of a model organism.—Four prop-erties that make fungi attractive for studies ofplasticity are (i) the ability to use clones forcomprehensive genetic investigations (i.e. the capac-ity to retain genotypes for multiple testing in differentenvironments), (ii) the phenotypic reversibility of thesame culture over time and after sequential culturing,(iii) the ability to use fungal species in evolutionarystudies, either by selection experiments (Scheiner2002) or in comparative tests of adaptive roles and(iv) the fact that many fungi have indeterminategrowth and thus provide the opportunity to study thefunction of genetic mosaics and the utility of dynamicboundaries (Peabody et al 2003).

Experimental investigations of basic environmentalsignals, such as temperature, nutrients, pH, osmoticpressure, water potential, light and their interactions,also are possible (Wickerham and Kurtzman 1975;Hallsworth and Magan 1996; Peabody et al 2003,2005; Bago et al 2004). In addition complex signals,such as those that are involved in symbiotic associa-tions, host immune mechanisms, virulence, patho-genesis and antigen-antibody interaction (i.e. resis-tance) can be used as the target for reaction norminvestigations (Lachke et al 2000, Bolker 2001,Saikkonen et al 2004, Macoris et al 2006).

Andrews (1998) discusses the fundamental differ-ence between modular organisms, such as bacteriaand fungi, and unitary organism with determinantstructures (e.g. mobile animals). His review empha-sized the potential insights that are possible fromthinking about architectural modules employed bymicroorganisms to survive, disperse, grow and repro-duce. Even though the phenotypic plasticity in fungiis common and well known among mycologists(Slutsky et al 1985, Andrews 1992) it is generallyunder appreciated.

Studies with A. pullulans.—Some mycologists con-sider A. pullulans to be a black yeast, while othersrefer to it as a yeast-like fungus. A. pullulans has been

FIG. 3. Colony of A. pullulans cultured on 0.5% (w/v)salicin (A), glycerol (B) or i-erythritol (C) 4 wk at 25 C(column 1), 4 wk at 9 C (column 2) and 12 wk at 9 C(column 3). Each photo is 5 cm square; bar: C2 5 1 cm.

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redefined by Zalar et al (2008). They define fourvarieties of the species (var. pullulans, var. melano-genum, var. subglaciale and var. namibiae) anddescribe them. These varieties are members of classAscomycota, order Dothideales, family Dothideaceae,and all are thought to be restricted to clonalreproduction. The versatile appetite and toleranceto stress of A. pullulans might explain its widespreadoccurrence in nature. It has been found in mosthabitats and is prominent in the phyllosphere. It isalso airborne (Shelton et al 2002, Samson et al 2004)and has been implicated as a major cause of allergicdisease in humans; recent studies on conidia emittedfrom flowers suggest high-molecular weight allergensin A. pullulans are responsible (Taylor et al 2005).Aureobasidium pullulans has been used for theproduction of extracellular compounds, especiallypolysaccharides such as pullulan (Catley 1979) andenzymes (Buzzini and Martini 2002). Its importanceto the biological control of plant diseases has receivedconsiderable attention (Buck et al 1998, Janisiewiczand Korsten 2002) and has become the modelorganism for ecological studies of the phylloplane(Andrews et al 2002, McGrath and Andrews 2007,Woody et al 2007).

A. pullulans is multiphasic and can undergo abudding to hyphal-form change. Its life cycle (Pechakand Crang 1977, Kockova-Kratochvılova et al 1980)involves the formation of blastoconidia that can giverise to promycelium that either can form (i) aerialhyphae that produce chlamydospores divided bysepta (also called arthroconidia) that bud to formblastoconidia or (ii) hyphae with lateral outgrowths ofblastococonidia that form chlamydospores with mel-anin in their cell walls (chlamydospores are freedfrom the hyphae and bud to form hyaline blastoco-nidia). Colony color in A. pullulans varies consider-ably among strains, and its expression is influenced bytemperature and pH (Wickerham and Kurtzman1975). Pollock et al (1992) obtained several strainsof A. pullulans from leaf surfaces and observedvariation in colony morphology, including a greatrange in colony color, extent of radial filamentousgrowth and degree of pigment accumulation. Inaddition they observed concentric rings of pigmenta-tion when colonies grew in light-dark cycles. Studieson mechanisms for changes in growth form fromyeast to mycelial (i.e. the YM growth transition) havereported the following interesting features.

Kockova-Kratochvılova et al (1980) illustrate themultiple growth forms of giant colonies that developfor A. pullulans. They diagram and describe diversecolors, varied margins, sectored growth, concentricgrowth stages and developmental patterns, all similarto what we find in our study. A diversity of other

investigations on the role of carbon and nitrogencompounds in artificial medium demonstrates pro-nounced affects on the YM transition (Kockova-Kratochvılova and Hronska 1981, Pasquier-Clouetand Zucca 1987). In some cases the compounds(e.g. alcohols) might function by induction ofchanges in the plasma membrane (Sevilla et al 1983;Moragues et al 1988, 1994) and induce filamentousgrowth (in S. cerevisae Lorenz et al 2000). Moreoverincreases in Zn2+ additions to continuous culturescaused a gradual shift through three growth regions(Reeslev et al 1993). Related to the YM-transition, thechange from yeast-like cells to chlamydospores isinfluenced by both glucose concentration and alimiting nitrogen source (Bermejo et al 1981) as wellas interstrain variability (Gadd and Cooper 1984).

Metabolic processes involved in the YM transitionare not well know but glutathione metabolism thatleads to reduced thiol might play a role in the YMtransition ( Jurgensen et al 2001) and t-RNA methyl-ation also might affect the differentiation process(Steiman et al 1990).

General observations on the change to mycelialgrowth include a decrease in both protein and RNAcontent (Sevilla et al 1988), the fact that hyphal andunicellular blastospores do not produce pullulan-likecompounds (Simon et al 1993, Campbell et al 2004)and that mycelial versus yeast development is afunction of population density (Park 1984, Finlay1987). It should be noted that hyphae becomemultinucleate as a result of the uncoupling of nucleardivision from cytokinesis. This change in the ploidylevel is largely due to changes in actin structure(Kopecka et al 2003).

Dimorphism in other fungi.—Early studies on theinfluences of external factors on dimorphism infungi, especially different growth forms of pathogenictaxa, were described by Gilchrist and Stokes (1898)and Ricketts (1901), who recognized the two mor-phological states of Blastomyces dermatitidis. Thesestudies led to experimental demonstration thatincubation temperature was important in the transi-tion between a ‘‘yeast-like’’ phase and a ‘‘mould-phase’’ (Hamburger 1907). In Rippon’s (1980)comprehensive review of dimorphism in pathogenicfungi he concluded, ‘‘The single most importantexternal factor affecting the growth phase of dimor-phic pathogenic fungi is temperature.’’ A review of260 publications (Bemmann 1981) on the dimor-phism of fungi summarizes these factors as influencesof dimorphism: atmosphere, temperature, inoculum,pH, carbon and energy sources as well as nitrogenand other supplements. Nickerson et al (2006)reviewed the phenomenon of quorum sensing in

SLEPECKY AND STARMER: PHENOTYPIC PLASTICITY IN FUNGI 827

dimorphic fungi and listed similar environmentalsignals that induce the YM transition. These includenutrient limits, pH, nitrogen source, CO2, chelatingagents and transition metals (Romano 1966). Inocu-lation size also influences the growth phase and isthought to be a general determinant of colonymorphology (Hornby et al 2004, Nickerson et al2006). This phenomenon is possibly due to quorumsensing (Hornby et al 2001) but is known only in a fewfungi (Candida albicans and Ceratocystis ulmi).

There is no doubt that signaling pathways andgenetic networks play a fundamental role in sensingenvironmental cues that induce the morphogenesisthat characterizes phenotypic plasticity in fungi(Madhani and Fink 1998, Lengeler et al 2000). Infact fungi can use similar signaling pathways inresponse to environmental signals that ultimatelyresult in same or even different outcomes. In a studyby Sanchez-Martınez and Perez-Martın (2001) threefungi (Candida albicans, Saccharomyces cerevisae andUstilago maydis) respond to nutrient limitation byusing the same signaling transduction pathways butproduce either pseudo-hyphal growth (C. albicansand S. cerevisae) or grow by budding (U. maydis).

The linkage of the YM transition with sexualreproduction and virulence apparently has an ancientorigin and is widespread in fungi (Madhani and Fink1998, Nadal et al 2008, Morrow and Fraser 2009).Virulence in human pathogenic fungi is induced bytemperature and accompanied by the phase transi-tion from the mycelial to the yeast growth form.Nemecek et al (2006) demonstrate that a hybridhistidine kinase senses the host signal and induces thephase transition that results in virulent gene expres-sion. In this case the kinase triggers a two-componentsignaling pathway. In general morphogenesis andvirulence is regulated by the conserved cAMP (cyclicadenosine monophosphate) and mitogen-activatedprotein (MAP) kinase signaling pathway in a variety offungi and is know to be involve in sensing nutrientlevels in S. cerevisae (Lee et al 2003, Breitkreutz et al2003, Xu 2000). Lee et al (2003) review the extensiveliterature and associated reviews that focus onphytopathogens. They implicate cAMP signaling inreproductive functions (mating, sporulation andspore germination) and other morphological transi-tions, such as hyphal development, infectious struc-ture and sclerotia formation. It is clear that the cAMPand MAP signaling is important to phenotypicplasticity in fungi and that two-component signalingis a common means for fungi to sense and react toenvironmental signals (Klein and Tebbets 2007).

Candida glabrata, a yeast that undergoes bothbudding-hyphal transitions and high-frequency-phe-notypic switching (both of which are thought to

contribute to successful pathogensis), is reversiblyswitched by regulation of MT-II metallothioneingenes and a hemolysin-like protein, HLP. It ishypothesized that switching is a means to achievephenotypic plasticity that lets populations respondrapidly to the changing physiology and immuneresponses of the host, as well as to antibiotictreatment (Lachke et al 2000). This adaptive expla-nation for high-frequency phenotypic switching hasbeen proposed as the mechanism used by otheropportunistic pathogens (C. albicans) that respondquickly to changes in their host (Soll 2002). At leastone antibiotic (Valinomycin) is known to suppresshyphal growth in C. albicans (Watanabe et al 2005).

Promislow (2005) used the expression level ofmRNA genes in S. cerevisae as the phenotype to study theeffects of environmental stressors on phenotypic plastic-ity. The plasticity of each gene was compared as to theirconnectivity to transcription factors and to downstreamgenes. He also studied the association of the gene withbiological processes to see whether the degree ofplasticity was related to their function. He found thefollowing: (i) The number of transcription factors thatregulate phenotypic plasticity was influenced by thesubstructure of the network in which it was associated;(ii) phenotypic plasticity is associated with a gene’sbiological function. (i.e. those genes involved in energyutilization, protein production and defense had highphenotypic plasticity, while those involved in DNAprocessing and transcription had low phenotypic plas-ticity); and (iii) phenotypic plasticity is correlated withthe number of transcription factors associated with agene.

Responses to light.—Responses to light (Marsh et al1959, Wenger and Lilly 1966, Bourret et al 1969) andcircadian rhythms appear to be widespread in fungalgroups. The capacity to anticipate regular environ-mental change by using circadian rhythms is largelybased on a conserved transcription-translation-basednegative feedback loop that can be reset by light andtemperature (Dunlap and Loros 2006). Circadianclock genes known in Neurospora crassa have homo-logs in several other fungi (Lombardi and Brody2005). Their survey included 17 fungal genomes anddiscovered that 11 well described clock controlledgenes (ccg) of Neurospora to be conserved in theother fungi. They found that a ccg-8 in Neurospora is ahomolog of opi1p (a gene involved in transcriptionalregulation of inositol in S. cerevisae). The lightresponse in A. pullulans found by Pollock et al(1992), which we report here for A. pullulans, is notknown to be under the influence of clock-controlledgenes, but the observation that growth ring formationcease after returning cultures to the dark suggests that

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the response might not be controlled by a clock-likemechanism.

Epigenetic effects.—The results reported here on thepotential epigenetic influences and the capacity todissect and understand the genetic pathways involvedin the YM transition suggest a promising direction forfuture research into the genetics and mechanismsinvolved in switching cell types and achieving mor-phological diversity from a single genotype. A.pullulans grows in the mycelial phase when dulcitol(galactitol) is the sole carbon source (FIG. 2); thisswitch can be suppressed by addition of nicotinamidebut not nicotinic acid. Nicotinamide is a potentinhibitor of sirtuins (Sir2) and changes in nicotin-amide concentration in the cell nucleus causesconcomitant changes in Sir2-mediated gene silencing.Silencing is a consequence of deacetylation of histonesthat lead to transcriptional down regulation (Sauve et al2006). In the experiments with A. pullulans reportedhere it is possible that nicotinamide is suppressinghistone deacetylases that modify chromatin structures.This could imply a change in the regulation of geneexpression (Prusty et al 2008), and thus the YMtransition is likely to be an epigenetic phenomenon.In fact in S. cerevisae Halme et al (2004) demonstratedthat epigenetic silencing of the gene FLO11 regulates akey developmental switch that controls growth of eitherpeudohyhae (FLO11 on) or yeast cell forms (FLO11silent). In addition Reyna-Lopez and Ruiz-Herrera(2004) showed that changes in DNA methylationpatterns occur during the dimorphic transitions ofMucor rouxii, Yarrowia liplolytica and Ustilago maydis.These results provide additional evidence that under-standing the epigenetic aspects of phenotypic plasticityis a worthwhile pursuit.

Conclusions.—Many of the above studies were con-cerned with changes in cell morphology, and theconnection to colony morphology is not alwaysestablished. However Park (1982) showed the Y/Mforms are influenced by the types of nitrogen in themedium and that M forms can develop within a Ycolony. We want to emphasize that the overall colonymorphology and the dynamics involved are also ofimportance and are diverse. This time-dependentcharacteristic could be of interest, especially in thecontext of modularity (Andrews 1998) and thepotential for understanding the mechanism involvedin achieving the diversity of colony forms.

The diversity of changes that are seen in A.pullulans (FIGS. 1, 2, 3) illustrate the spectrum ofthe reaction norm. In general the phenotypesobserved involve the entire population of cells andthus differ from phenotypic switching that spontane-ously occurs in a small proportion of the population

in Candida albicans and Cryptococcus neoformans(Slutsky et al 1985, Guerrero et al 2006); howeverthe sectoring observed (e.g. FIG. 1 glucose) could besimilar. Time-dependent changes (FIG. 3) that occurin concentric zones demonstrate synchronism inphenotypic changes as the environment changes.These changes are probably a result of nutrientdepletion as well as exudates of the colony as itspreads across the agar surface. This phenomenonprovides a unique opportunity to study time-depen-dent changes in gene expression that lead to changesin the phenotype.

Given that A. pullulans is ubiquitous, serves as amodel organism in the study of the phyllosphere andis important to both industry and medicine, we areenthusiastic about the potential use of this organismto investigate a diversity of problems on the proxi-mate and ultimate cause of phenotypic plasticity. Inthis case we do not know the molecular causes, butthe cascade of events induced by different environ-mental signals could lead to (i) different epigeneticinfluences, (ii) alteration of anabolic and catabolicprocesses, (iii) changes in cell morphology and thus(iv) different colony forms. Ultimately the adaptivefeatures of the colony form (e.g. dispersal, survivaland reproduction) will be instructive in understand-ing the evolution of multicellular phenotypes.

ACKNOWLEDGMENTS

We appreciate the technical assistance of Virginia Aber-deen, advice of Larry Wolf and Mike Cosgrove. The researchwas partially supported by the National Science Foundation.We thank Andre Lachance for providing the partial rDNAsequence.

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