Mycologia, 101(6), 2009, pp. 823832. DOI: 10.

3852/08-197
# 2009 by The Mycological Society of America, Lawrence, KS 66044-8897

Phenotypic plasticity in fungi: a review with observations on

Aureobasidium pullulans
Ralph A. Slepecky
William T. Starmer1

sources include glass (Schabereiter-Gurtner et al

2001), painted material (Shirakawa et al 2002), as
well as rocks and marble (Urz` et al 1999, 2001). It is
found in soil, freshwater and saltwater, ice (Zalar et al
2008) and is commonly recovered from the atmosphere (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). Unusual
sources 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 nuclear
reactors (Zhdanova et al 2000). Aureobasidium pullulans is involved as the principal colonizer initiating
biodeterioration (e.g. plasticized polyvinyl chloride,
Webb et al 2000) has been used as an indicator of
environmental pollution (Deshpande et al 1992) and
is implicated in human disease (Taylor et al 2005).
The pleomorphic characteristic of fungi (Savile
1969) is also know as phenotypic plasticity, that is
the ability of any organism to respond to environmental signals by altering morphology, physiological
state or behavior (West-Eberhard 1989). This ability is
widespread among taxa and has been studied
extensively primarily because of its importance to an
organisms ability to survive and propagate. The
function that describes the range of phenotypes
produced by a single genotype in a suite of
environments is called a reaction norm and is a
concept generally adopted by geneticists studying
evolution and ecology (Pigliucci 1996). Because over
their lifetimes organisms occur in changing environmental conditions they are expected to have reaction
norms that scale to the variable environment they
inhabit and thus the individual is expected to be
phenotypically plastic. What determines the shape of
the reaction norm and how the change from one
phenotype to another occurs are central questions in
molecular, evolutionary and ecological genetics.
Pigliucci (1996) discusses two broad approaches to
studying phenotypic plasticity. One is statistical, which
uses the tools developed by students of quantitative
genetics. The major limitation of this method is that
the assumptions underlying the theory are often too
simple and as a consequence inferences about genetic
mechanisms can be unrealistic. The second approach
is a mechanistic study of the genes involved in
phenotypic plasticity. The initial phase of this
approach is to use a genetic screen designed to

Department of Biology, Syracuse University, Syracuse,

New York 13244

Abstract: Phenotypic plasticity in fungi is reviewed in

the context of observations on phenotypic changes in
the colony morphology of the fungus Aureobasidium
pullulans. The variation in colony form is shown to
depend on (i) the types of single carbon substrates
(sugars and sugar alcohols) used in the growth
medium, (ii) colony age, (iii) incubation temperature, (iv) light cycle and (v) substrate type. Expanding
colonies grow in a developmental sequence that show
synchronize growth phase shifts as well as unusual
transitions from homogeneous to sectored, yeast to
mycelial and giant to microcolonial growth forms.
Epigenetic influences on phenotypic switches are
suggested to be potential causes of form changes. The
desirable properties of a model organism for studying
phenotypic plasticity are discussed and past work on
the yeast-mycelial transition of fungi is reviewed.
Key words: epigenetics, model organism, yeastmycelial transition
INTRODUCTION

Fungi are notable for their ability to switch growth

forms in response to environmental stimuli (Rayner
and Coates 1987). Most likely fungi rely on the
capacity to make these shifts to achieve survival,
dispersal and reproductive advantages, and no doubt
their success at these fundamental processes helps
explain their recognition as a kingdom. The ability of
fungi to alter forms and shift to different modes of
living has been of interest to mycologists because of
their importance to understanding fungal molecular
biology, ecology and evolution as well as their utility
in industry and their role in both infection and
biological control. One fungus that assumes many
different shapes (i.e. it is pleomorphic) and lives in a
wide variety of habitats is Aureobasidium pullulans.
This fungus has been recovered from diverse surfaces
types, especially the phylloplane (Andrews et al 2002,
McGrath and Andrews 2007, Andrews and Harris
1997, Woody et al 2007). Examples of other surface
Accepted for publication 7 May 2009.
1
Corresponding author. E-mail: wstarmer@syr.edu

823

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MYCOLOGIA

detect plasticity genes or genetic networks involved in

the transition from one phenotype to another. To
facilitate this type of work model plants and animals,
such as Arabidopsis thaliana or Drosophila melanogaster, often are employed as experimental organisms.
Even though microorganisms have been used for
studies in plasticity (e.g. Promislow 2005, Stomp et al
2008), their great potential for understanding the
mechanism of phenotypic plasticity have not been
generally recognized, especially in fungi ( Jennings
1993, Andrews 1992, Bago et al 2004). Bacterial
colonies growing on the surfaces in Petri dishes show
differentiated structures that result from a complex
series of morphological events. The geometry of
bacterial colonies can be a consequence of swarming,
chemotactic auto-aggregation, self-engineering, intercellular communication, nutrient gradients and stress
(Shapiro 1995, Ben-Jacob and Levine 2006). Shapiro
(1998) emphasized the need to consider a bacterial
population as a multicellular organism with complex
signaling systems that result in coordinated behaviors.
These emergent phenotypes of single cells growing
together and communicating affect survival, movement and reproduction, all of which can be beneficial
and thus evolve. Pattern formation in bacteria, such as
Bacillus subtilis (Mimura et al 2000), exemplify the
degree of phenotypic plasticity that can occur in
microorganisms grown in relatively simple culture
conditions. In this case colony morphology show
characteristics of phase transitions where there are
abrupt changes from one morphologic type to
another along nutrient and agar density gradients.
We report here that the fungus Aureobasidium
pullulans reversibly forms different types of colonies
depending on the substrate and temperature on
which it is grown, that is its environment. This
property coupled with other natural attributes suggests that this microorganism could serve as a model
for investigating a diversity of problems on the causes
of phenotypic plasticity. Along with our observations
on A. pullulans we discuss and review (i) the desirable
properties of a model organism, (ii) dimorphism in
fungi, (iii) responses to light and (iv) epigenetic
influences on phenotypic plasticity.
MATERIALS AND METHODS

The source of inocula was a single strain of A. pullulans (04313.1) isolated from beetles in morning glory flowers,
Massai Kopjes, Serengeti National Park, in 2004. Most of the
growth media were single-carbon sources in agar. A detailed
description of these media is given by Yarrow (1998). In
essence growth tests for carbon assimilation were conducted
on medium in 95 mm glass Petri dishes composed of 2%
Bacto agar, 0.67% YNB (Bacto yeast-nitrogen base) and
0.5% or 2.0% (w/v) carbon source. The initial inoculum

(,104 yeast-phase cells suspended in 1 mL H2O) was taken

from a culture growing on YNB glucose (0.5%) agar and was
placed on the center of the agar medium in the Petri dish.
The inoculated plates were sealed around the rims with
parafilm. Colonies were grown 23 d up to 34 mo at 9, 20
or 25 C. Photographs were taken under standard conditions
at various intervals over the period. Additional growth tests
were conducted under (i) constant light or dark and a
light : dark (12 h : 12 h) photoperiod, (ii) with inocula from
colonies growing on different sugar sources, (iii) using
different nitrogen sources added to YCB (Bacto yeastcarbon base) and (iv) conditions that potentially demonstrate epigenetic influences on changing colony morphology. Experiments in this latter category were conducted by
supplementing with 20 mM nicotinamide or 20 mM nicotinic acid in buffered (100 mM phosphate) medium.
Nicotinamide (the amide of nicotinic acid) is known to
inhibit histone deacetylases that are essential for epigenetic
regulation of gene expression (Prusty et al 2008). However
medium supplemented with nicotinic acid is not expected
to produce epigenetic changes, and thus morphological
shifts that occur on medium with nicotinamide but not on
medium containing nicotinic acid can be a useful indicator
of epigenetic control. The major strain in this study was
subjected to PCR amplification and rDNA sequencing as
described by Lachance et al (2005).
RESULTS

We provided examples of the diversity of morphological (colony) responses. Colony morphology is relatively uniform after 4 wk on 0.5% glucose at 25 C
(FIG. 1A). Growth at 9 C (FIG. 1C, D) produces
considerable sectoring (i.e. sectors with melanin)
and distinct stages of growth if allowed to grow 12 wk.
Although relatively minor there was a distinct change
in response to light (FIG. 1B) with daily growth rings
apparent as the colony expanded for 3 wk. This photo
response is immediately interrupted if the colony is
shielded from light and allowed to grow in complete
dark in the same incubator. We recorded the effect of
increasing the concentration of glucose 0.52%
(FIG. 1E). In this case there are manifest changes in
the growth pattern with discrete dynamic phases of
growth as the colony expands. We recorded the effect
of growth on 2% glucose supplemented with 20 mM
nicotinamide (FIG. 1F). The colony under these
conditions is more similar to the growth on the lower
concentration (i.e. 0.5% glucose, FIG. 1A) and might
indicate an epigenetic effect.
Colony morphology was a spreading black (melanin) branching structure after 4 wk on 0.5% dulcitol
at 25 C (FIG. 2A). Influences of temperature and time
were recorded (FIG. 2F, G). Growth at 9 C was
recorded (FIG. 2F, G), which indicated that little to
no melanin accumulates at low temperature after
4 wk and somewhat more melanin is present after

SLEPECKY AND STARMER: PHENOTYPIC PLASTICITY IN FUNGI

825

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 and
front illuminated). Colonies grown under continuous light
or dark on the same medium appear similar to the colony
in A. C and D 5 0.5% glucose at 9 C for 4 and 12 wk
respectively; E 5 2.0% glucose at 25 C, F 5 2.0% glucose at
25 C supplemented with 20 mM nicotinamide. Each photo
is 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 at
25 C, 2.0% glucose, supplemented with 20 mM nicotinamide and 20 mM nicotinic acid respectively. Colonies
grown at 25 C without supplements appear similar to the
colony in A. D and E 5 4 wk at 25 C, 0.5% glucose,
supplemented with 20 mM nicotinamide and 20 mM
nicotinic acid respectively. Colonies grown at 25 C without
supplements appear similar to the colony in A. F and G 5
0.5% glucose at 9 C for 4 and 12 wk respectively. Each photo
is 5 cm square; bar: A 5 1 cm.

12 wk. We also recorded growth on 0.5% (FIG. 2B, C)

and 2% (Fig. 2D, E) dulcitol respectively. Colonies
grown on media supplemented with 20 mM nicotinamide were recorded (FIGS. 2B, D), as were those
grown in media supplemented with 20 mM nicotinic
acid (FIGS. 2C, E). In this case an epigenetic effect is
implicated because the amide is expected to induce
changes in the chromatin while the acid is not and
should appear like the colony (FIG. 2A) on medium
that was not supplemented. Note that this is true for
either concentration of dulcitol.
Colony morphology was noted (FIG. 3) on other
carbon sources (0.5%, A 5 salicin, B 5 glycerol and C
5 i-erythritol) when incubated 4 wks at 25 C (column
1) and 4 wk at 9 C (column 2) or 12 wk at 9 C
(column 3). Lower temperature resulted in more
melanin and in some cases (glycerol and i-erythritol)
dynamic developmental phases of growth. These
examples illustrate the diverse and complex changes

that occur over time and as a consequence of

alterations in simple carbon sources.
The colonies (FIGS. 1, 2, 3) were grown from a
suspension of cells from a single strain of A. pullulans
grown on YNB + 0.5% glucose agar. Experiments
conducted with inocula from colonies grown on four
different carbon sources (0.5% w/v, glucose, dulcitol,
mannitol and salicin) each transferred to all four
original sources showed that morphological changes
experienced on the original plates were reversible.
Not all tests are shown, but other carbon and nitrogen
sources showed diverse phenotypes and changes over
time. Tests with other strains of A. pullulans
produced similar results. This was expected and
indicated that intraspecific variation in phenotypic
plasticity also exists. Partial rDNA sequence of the
strain (04-313.1, GenBank: FJ744598) in these studies
had strong similarity (greater than 98% similarity) to
many sequences from different strains of A. pullulans

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MYCOLOGIA
adhesive nets and conidial traps, and for mycoparasitism by developing hyhal coils and appressoria
(Nordbring-Hertz 2004). By far the most extensively
studied phenotypic change in fungi is the yeastmycelial (YM) transition. Several reviews of dimorphism in fungi discuss general features (Scherr and
Weaver 1953, Romano 1966, Bemmann 1981), phase
transitions and cell differentiation (Maresca and
Kobayashi 1989), signaling (Lengeler et al 2000, Lee
et al 2003), quorum sensing (Nickerson et al 2006),
regulation (Rayner and Coates 1987) and pathogenicity in plants and animals (Rippon 1980, Madhani
and Fink 1998, Lee et al 2003, Nadal et al 2008,
Morrow and Fraser 2009).

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.

deposited in GenBank. The strongest similarity (99%)

was with group 3 and 4 strains defined respectively as
A. pullulans var. subglaciale and var. namibiae by Zalar
et al (2008).
DISCUSSION

Colony changes due to mutagenesis were important

in early studies in microbial genetics. For example the
fact that DNA or its modification is the genetic
determinant was revealed by colony changes in
pneumococcus (Avery et al 1944). However phenotypic change (not due to treatment with mutagens) in
colonies is not often observed, possibly because pure
cultures have only a single characteristic colony
morphology or variation in colony type is seen only
among strains. However fungi are well known for
their extreme phenotypic plasticity (Slutsky et al 1985,
Andrews 1992, Bago et al 2004) and have properties
that make them suitable for laboratory studies of how
and why phenotypes change when environmental
conditions change. It is widely thought that fungi
develop differentially to explore and exploit the
environment and to respond quickly by changing
mycelia when environmental conditions change. For
example the nematode-trapping fungus Arthrobotrys
oligosopora develops different structures from mycelia
for infecting and parasitizing nematodes or other
fungi. Their response to environmental signals can
result in structures for catching nematodes, such as

Desirable properties of a model organism.Four properties that make fungi attractive for studies of
plasticity are (i) the ability to use clones for
comprehensive genetic investigations (i.e. the capacity to retain genotypes for multiple testing in different
environments), (ii) the phenotypic reversibility of the
same culture over time and after sequential culturing,
(iii) the ability to use fungal species in evolutionary
studies, either by selection experiments (Scheiner
2002) or in comparative tests of adaptive roles and
(iv) the fact that many fungi have indeterminate
growth and thus provide the opportunity to study the
function of genetic mosaics and the utility of dynamic
boundaries (Peabody et al 2003).
Experimental investigations of basic environmental
signals, such as temperature, nutrients, pH, osmotic
pressure, 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 associations, host immune mechanisms, virulence, pathogenesis and antigen-antibody interaction (i.e. resistance) can be used as the target for reaction norm
investigations (Lachke et al 2000, Bo lker 2001,
Saikkonen et al 2004, Macoris et al 2006).
Andrews (1998) discusses the fundamental difference between modular organisms, such as bacteria
and fungi, and unitary organism with determinant
structures (e.g. mobile animals). His review emphasized the potential insights that are possible from
thinking about architectural modules employed by
microorganisms to survive, disperse, grow and reproduce. Even though the phenotypic plasticity in fungi
is common and well known among mycologists
(Slutsky et al 1985, Andrews 1992) it is generally
under appreciated.
Studies with A. pullulans.Some mycologists consider A. pullulans to be a black yeast, while others
refer to it as a yeast-like fungus. A. pullulans has been

SLEPECKY AND STARMER: PHENOTYPIC PLASTICITY IN FUNGI

redefined by Zalar et al (2008). They define four
varieties of the species (var. pullulans, var. melanogenum, var. subglaciale and var. namibiae) and
describe them. These varieties are members of class
Ascomycota, order Dothideales, family Dothideaceae,
and all are thought to be restricted to clonal
reproduction. The versatile appetite and tolerance
to stress of A. pullulans might explain its widespread
occurrence in nature. It has been found in most
habitats and is prominent in the phyllosphere. It is
also airborne (Shelton et al 2002, Samson et al 2004)
and has been implicated as a major cause of allergic
disease in humans; recent studies on conidia emitted
from flowers suggest high-molecular weight allergens
in A. pullulans are responsible (Taylor et al 2005).
Aureobasidium pullulans has been used for the
production of extracellular compounds, especially
polysaccharides such as pullulan (Catley 1979) and
enzymes (Buzzini and Martini 2002). Its importance
to the biological control of plant diseases has received
considerable attention (Buck et al 1998, Janisiewicz
and Korsten 2002) and has become the model
organism 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 a
budding to hyphal-form change. Its life cycle (Pechak
and Crang 1977, Kockova-Kratochvlova et al 1980)
involves the formation of blastoconidia that can give
rise to promycelium that either can form (i) aerial
hyphae that produce chlamydospores divided by
septa (also called arthroconidia) that bud to form
blastoconidia or (ii) hyphae with lateral outgrowths of
blastococonidia that form chlamydospores with melanin in their cell walls (chlamydospores are freed
from the hyphae and bud to form hyaline blastoconidia). Colony color in A. pullulans varies considerably among strains, and its expression is influenced by
temperature and pH (Wickerham and Kurtzman
1975). Pollock et al (1992) obtained several strains
of A. pullulans from leaf surfaces and observed
variation in colony morphology, including a great
range in colony color, extent of radial filamentous
growth and degree of pigment accumulation. In
addition they observed concentric rings of pigmentation when colonies grew in light-dark cycles. Studies
on mechanisms for changes in growth form from
yeast to mycelial (i.e. the YM growth transition) have
reported the following interesting features.
Kockova-Kratochvlova et al (1980) illustrate the
multiple growth forms of giant colonies that develop
for A. pullulans. They diagram and describe diverse
colors, varied margins, sectored growth, concentric
growth stages and developmental patterns, all similar
to what we find in our study. A diversity of other

827

investigations on the role of carbon and nitrogen

compounds in artificial medium demonstrates pronounced affects on the YM transition (KockovaKratochvlova and Hronska 1981, Pasquier-Clouet
and Zucca 1987). In some cases the compounds
(e.g. alcohols) might function by induction of
changes in the plasma membrane (Sevilla et al 1983;
Moragues et al 1988, 1994) and induce filamentous
growth (in S. cerevisae Lorenz et al 2000). Moreover
increases in Zn2+ additions to continuous cultures
caused a gradual shift through three growth regions
(Reeslev et al 1993). Related to the YM-transition, the
change from yeast-like cells to chlamydospores is
influenced by both glucose concentration and a
limiting nitrogen source (Bermejo et al 1981) as well
as interstrain variability (Gadd and Cooper 1984).
Metabolic processes involved in the YM transition
are not well know but glutathione metabolism that
leads to reduced thiol might play a role in the YM
transition ( Jurgensen et al 2001) and t-RNA methylation also might affect the differentiation process
(Steiman et al 1990).
General observations on the change to mycelial
growth include a decrease in both protein and RNA
content (Sevilla et al 1988), the fact that hyphal and
unicellular blastospores do not produce pullulan-like
compounds (Simon et al 1993, Campbell et al 2004)
and that mycelial versus yeast development is a
function of population density (Park 1984, Finlay
1987). It should be noted that hyphae become
multinucleate as a result of the uncoupling of nuclear
division from cytokinesis. This change in the ploidy
level is largely due to changes in actin structure
(Kopecka et al 2003).
Dimorphism in other fungi.Early studies on the
influences of external factors on dimorphism in
fungi, especially different growth forms of pathogenic
taxa, were described by Gilchrist and Stokes (1898)
and Ricketts (1901), who recognized the two morphological states of Blastomyces dermatitidis. These
studies led to experimental demonstration that
incubation temperature was important in the transition between a yeast-like phase and a mouldphase (Hamburger 1907). In Rippons (1980)
comprehensive review of dimorphism in pathogenic
fungi he concluded, The single most important
external factor affecting the growth phase of dimorphic pathogenic fungi is temperature. A review of
260 publications (Bemmann 1981) on the dimorphism of fungi summarizes these factors as influences
of dimorphism: atmosphere, temperature, inoculum,
pH, carbon and energy sources as well as nitrogen
and other supplements. Nickerson et al (2006)
reviewed the phenomenon of quorum sensing in

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MYCOLOGIA

dimorphic fungi and listed similar environmental

signals that induce the YM transition. These include
nutrient limits, pH, nitrogen source, CO2, chelating
agents and transition metals (Romano 1966). Inoculation size also influences the growth phase and is
thought to be a general determinant of colony
morphology (Hornby et al 2004, Nickerson et al
2006). This phenomenon is possibly due to quorum
sensing (Hornby et al 2001) but is known only in a few
fungi (Candida albicans and Ceratocystis ulmi).
There is no doubt that signaling pathways and
genetic networks play a fundamental role in sensing
environmental cues that induce the morphogenesis
that characterizes phenotypic plasticity in fungi
(Madhani and Fink 1998, Lengeler et al 2000). In
fact fungi can use similar signaling pathways in
response to environmental signals that ultimately
result in same or even different outcomes. In a study
by Sanchez-Martnez and Perez-Martn (2001) three
fungi (Candida albicans, Saccharomyces cerevisae and
Ustilago maydis) respond to nutrient limitation by
using the same signaling transduction pathways but
produce either pseudo-hyphal growth (C. albicans
and S. cerevisae) or grow by budding (U. maydis).
The linkage of the YM transition with sexual
reproduction and virulence apparently has an ancient
origin and is widespread in fungi (Madhani and Fink
1998, Nadal et al 2008, Morrow and Fraser 2009).
Virulence in human pathogenic fungi is induced by
temperature and accompanied by the phase transition from the mycelial to the yeast growth form.
Nemecek et al (2006) demonstrate that a hybrid
histidine kinase senses the host signal and induces the
phase transition that results in virulent gene expression. In this case the kinase triggers a two-component
signaling pathway. In general morphogenesis and
virulence is regulated by the conserved cAMP (cyclic
adenosine monophosphate) and mitogen-activated
protein (MAP) kinase signaling pathway in a variety of
fungi and is know to be involve in sensing nutrient
levels in S. cerevisae (Lee et al 2003, Breitkreutz et al
2003, Xu 2000). Lee et al (2003) review the extensive
literature and associated reviews that focus on
phytopathogens. They implicate cAMP signaling in
reproductive functions (mating, sporulation and
spore germination) and other morphological transitions, such as hyphal development, infectious structure and sclerotia formation. It is clear that the cAMP
and MAP signaling is important to phenotypic
plasticity in fungi and that two-component signaling
is a common means for fungi to sense and react to
environmental signals (Klein and Tebbets 2007).
Candida glabrata, a yeast that undergoes both
budding-hyphal transitions and high-frequency-phenotypic switching (both of which are thought to

contribute to successful pathogensis), is reversibly

switched by regulation of MT-II metallothionein
genes and a hemolysin-like protein, HLP. It is
hypothesized that switching is a means to achieve
phenotypic plasticity that lets populations respond
rapidly to the changing physiology and immune
responses of the host, as well as to antibiotic
treatment (Lachke et al 2000). This adaptive explanation for high-frequency phenotypic switching has
been proposed as the mechanism used by other
opportunistic pathogens (C. albicans) that respond
quickly to changes in their host (Soll 2002). At least
one antibiotic (Valinomycin) is known to suppress
hyphal growth in C. albicans (Watanabe et al 2005).
Promislow (2005) used the expression level of
mRNA genes in S. cerevisae as the phenotype to study the
effects of environmental stressors on phenotypic plasticity. The plasticity of each gene was compared as to their
connectivity to transcription factors and to downstream
genes. He also studied the association of the gene with
biological processes to see whether the degree of
plasticity was related to their function. He found the
following: (i) The number of transcription factors that
regulate phenotypic plasticity was influenced by the
substructure of the network in which it was associated;
(ii) phenotypic plasticity is associated with a genes
biological function. (i.e. those genes involved in energy
utilization, protein production and defense had high
phenotypic plasticity, while those involved in DNA
processing and transcription had low phenotypic plasticity); and (iii) phenotypic plasticity is correlated with
the number of transcription factors associated with a
gene.
Responses to light.Responses to light (Marsh et al
1959, Wenger and Lilly 1966, Bourret et al 1969) and
circadian rhythms appear to be widespread in fungal
groups. The capacity to anticipate regular environmental change by using circadian rhythms is largely
based on a conserved transcription-translation-based
negative feedback loop that can be reset by light and
temperature (Dunlap and Loros 2006). Circadian
clock genes known in Neurospora crassa have homologs in several other fungi (Lombardi and Brody
2005). Their survey included 17 fungal genomes and
discovered that 11 well described clock controlled
genes (ccg) of Neurospora to be conserved in the
other fungi. They found that a ccg-8 in Neurospora is a
homolog of opi1p (a gene involved in transcriptional
regulation of inositol in S. cerevisae). The light
response in A. pullulans found by Pollock et al
(1992), which we report here for A. pullulans, is not
known to be under the influence of clock-controlled
genes, but the observation that growth ring formation
cease after returning cultures to the dark suggests that

SLEPECKY AND STARMER: PHENOTYPIC PLASTICITY IN FUNGI

the response might not be controlled by a clock-like
mechanism.
Epigenetic effects.The results reported here on the
potential epigenetic influences and the capacity to
dissect and understand the genetic pathways involved
in the YM transition suggest a promising direction for
future research into the genetics and mechanisms
involved in switching cell types and achieving morphological diversity from a single genotype. A.
pullulans grows in the mycelial phase when dulcitol
(galactitol) is the sole carbon source (FIG. 2); this
switch can be suppressed by addition of nicotinamide
but not nicotinic acid. Nicotinamide is a potent
inhibitor of sirtuins (Sir2) and changes in nicotinamide concentration in the cell nucleus causes
concomitant changes in Sir2-mediated gene silencing.
Silencing is a consequence of deacetylation of histones
that lead to transcriptional down regulation (Sauve et al
2006). In the experiments with A. pullulans reported
here it is possible that nicotinamide is suppressing
histone deacetylases that modify chromatin structures.
This could imply a change in the regulation of gene
expression (Prusty et al 2008), and thus the YM
transition is likely to be an epigenetic phenomenon.
In fact in S. cerevisae Halme et al (2004) demonstrated
that epigenetic silencing of the gene FLO11 regulates a
key developmental switch that controls growth of either
peudohyhae (FLO11 on) or yeast cell forms (FLO11
silent). In addition Reyna-Lopez and Ruiz-Herrera
(2004) showed that changes in DNA methylation
patterns occur during the dimorphic transitions of
Mucor rouxii, Yarrowia liplolytica and Ustilago maydis.
These results provide additional evidence that understanding the epigenetic aspects of phenotypic plasticity
is a worthwhile pursuit.
Conclusions.Many of the above studies were concerned with changes in cell morphology, and the
connection to colony morphology is not always
established. However Park (1982) showed the Y/M
forms are influenced by the types of nitrogen in the
medium and that M forms can develop within a Y
colony. We want to emphasize that the overall colony
morphology and the dynamics involved are also of
importance and are diverse. This time-dependent
characteristic could be of interest, especially in the
context of modularity (Andrews 1998) and the
potential for understanding the mechanism involved
in achieving the diversity of colony forms.
The diversity of changes that are seen in A.
pullulans (FIGS. 1, 2, 3) illustrate the spectrum of
the reaction norm. In general the phenotypes
observed involve the entire population of cells and
thus differ from phenotypic switching that spontaneously occurs in a small proportion of the population

829

in Candida albicans and Cryptococcus neoformans

(Slutsky et al 1985, Guerrero et al 2006); however
the sectoring observed (e.g. FIG. 1 glucose) could be
similar. Time-dependent changes (FIG. 3) that occur
in concentric zones demonstrate synchronism in
phenotypic changes as the environment changes.
These changes are probably a result of nutrient
depletion as well as exudates of the colony as it
spreads across the agar surface. This phenomenon
provides a unique opportunity to study time-dependent changes in gene expression that lead to changes
in the phenotype.
Given that A. pullulans is ubiquitous, serves as a
model organism in the study of the phyllosphere and
is important to both industry and medicine, we are
enthusiastic about the potential use of this organism
to investigate a diversity of problems on the proximate and ultimate cause of phenotypic plasticity. In
this case we do not know the molecular causes, but
the cascade of events induced by different environmental signals could lead to (i) different epigenetic
influences, (ii) alteration of anabolic and catabolic
processes, (iii) changes in cell morphology and thus
(iv) different colony forms. Ultimately the adaptive
features of the colony form (e.g. dispersal, survival
and reproduction) will be instructive in understanding the evolution of multicellular phenotypes.
ACKNOWLEDGMENTS

We appreciate the technical assistance of Virginia Aberdeen, advice of Larry Wolf and Mike Cosgrove. The research
was partially supported by the National Science Foundation.
We thank Andre Lachance for providing the partial rDNA
sequence.

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