Copyright  2001 by the Genetics Society of America
An STE12 Homolog From the Asexual, Dimorphic Fungus
Penicillium marneffei Complements the Defect in Sexual
Development of an Aspergillus nidulans steA Mutant
Anthony R. Borneman, Michael J. Hynes and Alex Andrianopoulos
Department of Genetics, University of Melbourne, Victoria, 3010 Australia
ABSTRACT
Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species
identified in its genus. At 25⬚ P. marneffei exhibits true filamentous growth, while at 37⬚ P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the
STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number
of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly
conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12␣ genes, form
a distinct subclass of STE12 homologs that have a C2H2 zinc-finger motif in addition to the homeobox
domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number
of mutants in the P. marneffei - type strain and, in combination with selectable markers, developed a highly
efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene
had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei.
Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of
an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias
against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified
cryptic sexual cycle.
S
IGNAL transduction pathways play a crucial role in
linking genetically programmed cellular events to
external stimuli in eukaryotic organisms. This is
achieved through protein-mediated transduction of signals from the cell membrane to the nucleus, which
ultimately leads to changes in gene expression. The
most common and intensively studied signaling pathway
is the mitogen-activated protein kinase (MAPK) pathway, which has been shown to be conserved throughout
eukaryotes including fungi, flies, worms, and humans.
In each of these organisms the MAPK module consists
of a phosphorylation cascade of three protein kinases,
a MAP kinase kinase kinase (MAPKKK), a MAP kinase
kinase (MAPKK), and a MAPK. The yeast Saccharomyces
cerevisiae contains some of the best-characterized MAPK
pathways and these have been shown to regulate a variety of processes including growth in high osmolarity
environments, cell integrity, spore formation, mating,
and pseudohyphal/filamentous growth (for a review see
Gustin et al. 1998). It has also been shown that the
activation of the MAPK modules of each of these pathways leads to the phosphorylation-mediated regulation
of specific downstream transcription factors to alter
gene expression (Cooper 1994; Marshall 1994; Cobb
and Goldsmith 1995).
Ste12p is the direct target of the MAPK for both the
Corresponding author: Alex Andrianopoulos, Department of Genetics,
University of Melbourne, Victoria, 3010 Australia.
E-mail: [email protected]
Genetics 157: 1003–1014 (March 2001)
mating and pseudohyphal growth pathways. These two
pathways share their MAPKKK (Ste7p) and MAPKK
(Ste11p) proteins but the MAPK of each cascade is
unique with Fus3p regulating Ste12p function in response to mating pheromone and Kss1p in response to
pseudohyphal signals (Cook et al. 1997; Madhani et al.
1997; Madhani and Fink 1998). Specificity of Ste12p
activation is achieved despite receiving signals from two
different pathways through combinatorial activation of
target genes. In response to pheromone, Ste12p homodimers or Ste12p-Mcm1p heterodimers activate transcription from PREs (pheromone response elements)
to induce the mating response (Kronstad et al. 1987;
Hagen et al. 1991; Song et al. 1991). During pseudohyphal growth, Ste12p requires the presence of a second,
filamentation-specific transcription factor, Tec1p, for
activation from FREs ( f ilamentation response elements) found upstream of genes responsible for pseudohyphal development (Gavrias et al. 1996; Madhani
and Fink 1997).
The study of STE12 homologs in other fungi has
shown that this regulator has been conserved at both
the sequence and functional levels. STE12 homologs
identified in the dimorphic yeast Candida lusitaniae
(CLS12) and the filamentous fungus Aspergillus nidulans
(steA) have been shown to regulate the mating response
without affecting any other cellular processes (Vallim
et al. 2000; Young et al. 2000). This phenotype is specific
to sexual development such that the A. nidulans steA
mutant fails to complete sexual reproduction, but is un-
1004
A. R. Borneman, M. J. Hynes and A. Andrianopoulos
affected in asexual reproduction (conidiation; Vallim
et al. 2000). STE12 homologs have also been identified
from two pathogenic species of fungi, Cryptococcus neoformans and Candida albicans, where Ste12p regulates
processes other than mating. In C. neoformans, an STE12
homolog is found only in the MAT␣ mating type of the
fungus and, while largely dispensable for mating, Ste12␣
is required for haploid fruiting and regulates virulence
depending on the serotype under investigation (Wickes
et al. 1997; Yue et al. 1999; Chang et al. 2000). C. albicans
has been proposed to be an asexual diploid yeast that
would not require genes involved in mating. However,
mating-type loci have recently been described in C. albicans and mating between strains of different mating types
demonstrated (Hull et al. 2000; Magee and Magee
2000). In C. albicans, the STE12 homolog CPH1 is required for hyphal growth on solid media and, in combination with a second developmental regulator, Efg1p is
required for virulence of C. albicans in an animal host
(Liu et al. 1994; Lo et al. 1997). The function of CPH1
in the cryptic sexual cycle has yet to be determined,
however.
Penicillium marneffei is an asexual ascomycete that displays a temperature-dependent dimorphic growth
switch (Garrison and Boyd 1973; Chan and Chow
1990). Unlike dimorphic fungi such as C. albicans and
C. neoformans, which are primarily yeast-like with a relatively minor filamentous form, P. marneffei is predominantly a filamentous species that has a significant unicellular growth form. As discussed above, STE12 homologs
have been shown to regulate filamentous growth associated with yeast-hyphal dimorphism in S. cerevisiae, C.
albicans, and C. neoformans. We therefore wished to analyze the function of an STE12 homolog in P. marneffei
to determine if, like the STE12 homologs in these species, it plays a role during dimorphic development in a
species that is primarily filamentous.
We have cloned the P. marneffei STE12 homolog stlA
and it is predicted to encode a protein that has two
potential DNA-binding domains: a homeobox domain
and a C2H2 Zn2⫹ finger motif. A DNA-mediated transformation system was developed for P. marneffei to allow
disruption of the stlA locus. The stlA deletion strain
displayed no detectable mutant phenotype despite the
high degree of stlA sequence conservation observed.
The stlA gene was shown to be functional in A. nidulans,
however, because it was able to complement the mating
defect of an A. nidulans steA deletion strain.
MATERIALS AND METHODS
Fungal strains and media: P. marneffei and A. nidulans strains
were grown on either Aspergillus nitrogen-free medium
(ANM; Cove 1966) supplemented with either 10 mm ␥-amino
butyric acid (GABA), sodium nitrate (NaNO3), or ammonium
tartrate (NH4T) as a sole nitrogen source, S. cerevisiae synthetic
dextrose (SD) medium (Ausubel et al. 1994), or brain heart
infusion (BHI) broth (Oxoid). Media for protoplast regenera-
tion contained 1.2 m sucrose as an osmotic stabilizer. The
P. marneffei - type strain FRR2161 was obtained from Dr. J. Pitt
(CSIRO Food Industries, Sydney). Strain SPM3 was isolated
as a FRR2161 sector resistant to chlorate, unable to grow on
nitrate as a sole nitrogen source, and presumed to be a nitrate
reductase mutant on the basis of previously defined growth
tests (Cove 1972). Strain SPM4 was isolated as a SPM3 sector
resistant to 1 mg/ml 5-fluoroorotic acid (5-FOA) that was
unable to grow in the absence of uridine (5 mm) and uracil
(5 mm). A. nidulans strain UI139 (biA1; ⌬argB; ⌬steA::argB)
was obtained from Dr. B. Miller (University of Idaho; Vallim
et al. 2000).
Protoplast transformation: The transformation procedure
for P. marneffei germlings is based on previously described
methods for A. nidulans transformation (Tilburn et al. 1983;
Yelton et al. 1984). Briefly, ⵑ1 ⫻ 108 P. marneffei spores were
harvested from ANM ⫹ GABA solid medium, inoculated into
400 ml of appropriately supplemented SD broth, and incubated at 37⬚ for 40 hr. Highly branched germlings were isolated by filtration through Miracloth (Calbiochem, La Jolla,
CA) and washed with 0.6 m MgSO4. Approximately 5 g wet
weight tissue was resuspended in 10 ml of chilled osmotic
buffer (1.2 m MgSO4, 10 mm NaOP, pH 5.8) and placed on
ice. Lytic enzyme (Sigma, St. Louis) and bovine serum albumin
were added to final concentrations of 5 mg/ml and 1.2 mg/ml,
respectively, and the mixture incubated for 1 hr at 30⬚ with
gentle agitation. Protoplasts were harvested and processed
according to the method previously described for the transformation of A. nidulans (Andrianopoulos and Hynes 1988).
Osmotically stabilized selection plates for SPM4 contained
either 10 mm NH4T as the sole nitrogen source (selecting for
pyrG complementation), 10 mm NaNO3, and 5 mm uridine
and uracil (selecting for niaD complementation), or 1.5 ␮g/
ml bleomycin, 10 mm NH4T, and 5 mm uridine and uracil
selecting for bleomycin resistance. A. nidulans protoplast transformations were performed as described previously using 1 ␮g/
ml bleomycin (final concentration) in the selection plates (Andrianopoulos and Hynes 1988).
Molecular techniques: The plasmids used in this study are
listed in Table 1. DNA for transformation was isolated using
either the Qiafilter 100 kit (QIAGEN, Chatsworth, CA) or the
High Purity Plasmid kit (Roche). To isolate genomic DNA,
100 ml of SD medium was inoculated with ⵑ1 ⫻ 106 P. marneffei
spores and incubated at 37⬚ for 40 hr. Germlings were isolated
by filtration through Miracloth (Calbiochem), washed with
water, blotted to remove excess fluid, and stored at ⫺20⬚.
Genomic DNA was prepared as described previously (Lee and
Taylor 1990). RNA was isolated from P. marneffei cultures
using the RNA Red fast prep kit (BIO 101, Vista, CA). For
the 25⬚ vegetative samples, FRR2161 conidia were inoculated
into SD medium and incubated at 25⬚ for 2 days with shaking
and mycelia harvested by filtration through Miracloth (Calbiochem). Conidiating cultures were prepared by filtering the
25⬚ vegetative cultures onto Whatman paper circles after 2
days of growth and placing the filters onto 0.1% glucose,
ANM ⫹ GABA agar plates for 4 days to allow for the production
of conidiophores. Yeast cultures were grown by inoculating
BHI (Oxoid) liquid medium with FRR2161 conidia and
allowing growth to proceed at 37⬚ with shaking for 4 days. At
this time, there was a mixture of both yeast cells and hyphal
filaments. A sample of the supernatant was taken after the
hyphal material was allowed to settle and transferred to new
medium for an additional 2 days of growth. Cells were harvested by filtration onto Whatman paper. After harvesting, all
samples were immediately frozen at ⫺70⬚. RNA was subjected
to gel electrophoresis on 1.2% agarose, formaldehyde denaturing gels. Southern and Northern blotting were performed
using H bond N⫹ membrane (Amersham) according to man-
An STE12 Homolog from P. marneffei
1005
TABLE 1
Plasmids used in this study
Plasmid
pJR15
pSTA14
pRS31
pAmPh520
pAB4342
pAB4458
pAB4623
pAB4624
pAB4625
pSTE12 2B
a
Features
Reference
A. nidulans pyrG genea
A. oryzae niaD gene
gpdA(p)::stuA::gfp
Tn5 ble R gene expressed from the N. crassa am promoter
1.4-kb XhoI-BamHI fragment of pJR15 end filled with
Klenow polymerase and inserted into the SmaI site of
pBluescript II SK⫹
3.4-kb EcoRI-Sal I genomic fragment in pBluescript II SK⫹
4.1-kb XbaI-Bgl II genomic fragment in pLitmus 29
4.1-kb XbaI-Bgl II fragment of pAB4623 cloned into the XbaI
and Bgl II sites of pAB4458
1.4-kb EcoRV-BamHI fragment of pAB4342 cloned into the
SmaI and Bgl II sites of pAB4625
A. nidulans steA genomic clone
Oakley et al. (1987)
Unkles et al. (1989)
Suelmann et al. (1997)
Austin et al. (1990)
This study
This study
This study
This study
This study
Vallim et al. (2000)
Obtained from the Fungal Genetics Stock Centre (Kansas).
ufacturer’s instructions. Filters were hybridized with [␣-32P]
dATP-labeled probes (random primer) and processed using
standard procedures (Sambrook et al. 1989).
Cloning and disruption of the stlA locus: The stlA gene was
isolated by degenerate PCR using the primers STE1 (GAARA
ARTTYGARGARGGNRT) and STE2 (AAIACYTTYTGYTTYT
TYTGNGT), designed using the conserved homeodomain regions of S. cerevisiae (X16112), C. albicans (L16451), Kluyveromyces lactis (L21156), and C. neoformans STE12 (AF012924)
homologs. PCR conditions consisted of an initial denaturation
step of 94⬚ for 2 min after which Taq DNA polymerase was
added and 30 cycles of 94⬚ for 30 sec, 45⬚ for 30 sec, and 72⬚
for 30 sec were performed. One broad band of ⵑ125 bp
in size was observed. Sequence analysis of individual clones
revealed that this band was composed of two major species,
one of which had significant homology to STE12 homologs.
This PCR product was used to probe a 3- to 4-kb EcoRI-Sal I
size-selected FRR2161 genomic library in pBluescript II SK⫹
(Stratagene, La Jolla, CA). One positive clone (pAB4458) that
contained the entire region shown to hybridize to the PCR
probe, but which lacked the 5⬘ portion of the gene, was isolated. A 4.1-kb XbaI-Bgl II fragment that overlapped the 5⬘ end
of pAB4458 was obtained from a ␭-GEM11 FRR2161 genomic
library and cloned into pLitmus 29 (New England Biolabs,
Beverly, MA) to give pAB4623. The XbaI-Bgl II fragment of
pAB4623 was cloned into the XbaI-Bgl II sites of pAB4458 to
give the full-length clone pAB4624. To disrupt stlA, a 1.4-kb
EcoRV-BamHI fragment containing the pyrG cassette of
pAB4342 was cloned into the SmaI-Bgl II sites of pAB4624 to
give pAB4625. pAB4625 was digested at the unique ApaI site
in the pBluescript II SK⫹ polylinker and subjected to gel electrophoresis, and the single digested band gel purified using
the Bresaclean Gel purification kit (Geneworks). A total of
500 ng of digested vector was transformed into SPM4 and
transformants selected for complementation of the uridine/
uracil auxotrophy.
Microscopy and cellular staining: P. marneffei was grown on
microscope slides coated with thin layers of either ANM ⫹
GABA or SD solid medium and incubated at 25⬚ or 37⬚ as
indicated (Borneman et al. 2000). Slides were viewed on a
Reichart Jung Polyvar II microscope using either differential
interference contrast (DIC) or epifluorescence optics to detect green fluorescent protein (GFP) fluorescence (band pass
450–495 nm, dichroic mirror 510 nm, barrier filter 520 nm).
Microscope images were captured with a SPOT CCD camera
(Diagnostic Instruments, Sterling Heights, MI) and processed
using Adobe Photoshop 4.0.
Complementation of the A. nidulans steA mutation: Strain
UI139 (⌬steA; Vallim et al. 2000) was cotransformed with 1.5
␮g of pAB4624 (stlA) and 1 ␮g of pAmPh520 (bleR; Austin
et al. 1990). Colonies resistant to 1 ␮g/ml bleomycin were
isolated and screened for their ability to self-fertilize. Spores
from single transformant colonies were stab inoculated onto
ANM ⫹ NO3 media and allowed to grow for 2 days at 37⬚.
Plates were sealed to promote the sexual cycle and examined
for the presence of sexual structures (cleistothecia) using a
dissection microscope after 7 days growth at 37⬚.
Analysis of stlA conservation: Pairwise protein and DNA
alignments were performed using GAP from the Wisconsin
Package (Deveraux et al. 1984) on the Australian National
Genomics Information Service. For the synonymous/nonsynonymous rate analysis, minor manual modifications were
made to the DNA alignments to optimize the alignments and
ensure codons were not split by gaps. Estimates of synonymous
and nonsynonymous substitution rates were made using the
YN00 program from the PAML package (Yang and Neilsen
2000). The YN00 algorithm takes into account both transition/transversion rate bias and base/codon frequency bias.
RESULTS
P. marneffei has an STE12 homolog: Degenerate PCR
primers were designed to the conserved homeodomain
encoding region of the S. cerevisiae STE12, C. albicans
CPH1, and C. neoformans STE12␣ genes. This PCR produced a fragment of 125 bp, which was shown by sequence analysis to be homologous to STE12. This product was used to probe P. marneffei genomic libraries (see
materials and methods), resulting in the isolation of
a full-length genomic clone, pAB4624. Sequencing of
this clone revealed an open reading frame (ORF), interrupted by four introns with extensive similarity to STE12,
and was designated stlA (sterile twelve like; accession
no. AF284062). GenBank database searches (BLAST)
1006
A. R. Borneman, M. J. Hynes and A. Andrianopoulos
Figure 1.—The stlA gene of P. marneffei encodes
a homeodomain and C2H2 Zn2⫹ finger domain
protein. (A) The restriction map of the stlA genomic locus and diagrammatic representation of the
predicted position of the coding region (black
boxes) separated by four introns. The position of
the homeodomain (hatched box) and the two
C2H2 Zn2⫹ fingers (open boxes) is indicated. The
region of DNA that is predicted to encode a sucrose transporter homolog is also indicated
(shaded box). (B) Alignment of the predicted
homeodomain of StlA with several other Ste12
homologs: A. nidulans SteA (AnSteA), C. neoformans
Ste12␣ (CnSte12␣), S. cerevisiae Ste12p (ScSte12),
K. lactis Ste12p (KlSte12), and C. albicans Cph1p
(CaCph1). The regions predicted to form the
three ␣-helices of the homeodomain are indicated by thick black lines. (C) Alignment of the
C2H2 Zn2⫹ fingers identified in the A. nidulans, C.
neoformans, and P. marneffei STE12 homologs. The
cysteine and histidine residues predicted to comprise the Zn2⫹ coordinating residues of the C2H2
fingers are indicated by an asterisk.
also identified a second ORF in this clone that had
significant identity to the SUC1 sucrose transporter of
Arabidopsis thaliana (S38197) and a putative sucrose
transporter from Schizosaccharomyces pombe (CAB16264)
(Figure 1A). On the basis of the gene structure of
the A. nidulans steA gene, stlA is predicted to encode a
689-amino-acid protein with significant similarity to all
identified STE12 homologs. This similarity is primarily
restricted to the region predicted to encode the homeobox DNA-binding motif common to all the Ste12 proteins (Figure 1B). StlA shows the highest degree of similarity to SteA of A. nidulans (71% identity and 84%
similarity) and Ste12␣ of C. neoformans (58% similarity
and 39% identity). In addition to the homeodomain
motif, these three proteins contain two C2H2 Zn2⫹ finger
domains (Figure 1C). This second putative DNA-binding motif is absent from the S. cerevisiae, K. lactis, and
C. albicans Ste12p proteins. The P. marneffei, A. nidulans,
and C. neoformans proteins also lack homology to the
regions of Ste12p that have previously been shown to
be important for regulation by Dig1p and Dig2p (Ste12p
residues 305–311) as well as the tyrosine residues (residues 307 and 314) required for repression of transcription by Ste12p in the absence of MAPK activation (Pi
et al. 1997).
The promoter of the stlA gene was examined for recognition sequences of known fungal developmental regulators to attempt to infer the pattern of regulation
expected for the stlA transcript. There are no sequences
present in the stlA promoter region that match the consensus of a S. cerevisiae PRE (Yuan and Fields 1991) or
FRE (Madhani and Fink 1997). A number of putative
binding sites were identified for regulators of asexual
development in A. nidulans, however, with six BrlA consensus binding sites (Chang and Timberlake 1993),
four AbaA sites (Andrianopoulos and Timberlake
1994), and one StuA site (Dutton et al. 1997) identified,
the majority of which are within 1 kb of the translational
start of stlA.
stlA is expressed primarily in vegetative tissues: The
An STE12 Homolog from P. marneffei
Figure 2.—stlA is expressed in vegetative tissue. RNA was
isolated from P. marneffei vegetative hyphal cells grown at 25⬚,
filamentous hyphal cells induced to undergo asexual development (25⬚C dev), and yeast cells grown at 37⬚ (see materials
and methods). Northern blots were hybridized with probes
specific for stlA to examine expression levels or A. nidulans
histone H3 (Ehinger et al. 1990) as a loading control.
data from the promoter analysis suggested that stlA may
be developmentally regulated, most likely being upregulated during conidiation due to the action of BrlA,
AbaA, and StuA. This pattern is observed in A. nidulans,
with expression of steA being almost undetectable in
vegetative tissues, but expressed at high levels upon the
commencement of asexual development despite SteA
not being required for conidiation (Vallim et al. 2000).
In S. cerevisiae, STE12 expression is 5- to 10-fold higher
in a or ␣ haploids than in a/␣ diploids (Fields and
Herskowitz 1987). Therefore, upregulation of STE12
expression appears to be correlated with induction of
the pathways that it regulates. Northern analysis was
performed to determine if any correlations between
stlA expression and developmental stages in P. marneffei
could be found. In P. marneffei the stlA gene produces
a single transcript of 2.8 kb that is present during both
filamentous and yeast growth states (Figure 2). The stlA
transcript is abundant in the vegetative filamentous
form, with expression decreasing to ⵑ40% of the vegetative level during conidiation. The level of stlA transcript
in the yeast form of P. marneffei was approximately equivalent to that observed in the vegetative filamentous
form. Therefore the expression of stlA is opposite to that
of the A. nidulans steA gene and suggests that, instead of
functioning after asexual development, stlA may function during vegetative growth. The decrease in stlA expression levels during conidiation may reflect a lack of
stlA expression in the developing conidiophore, because
asexual developmental cultures contain a mixture of
vegetative and developmental tissue types.
Isolation of pyrG and niaD mutant strains and transformation of P. marneffei: To examine the function of
the stlA gene in P. marneffei, it was necessary to develop
a DNA-mediated transformation procedure and to establish targeted gene deletion strategies. One of the
key elements of such procedures is suitable selectable
markers to allow identification of transformed strains.
P. marneffei orotidine 5⬘-monophosphate decarboxylase
1007
(pyrG) and nitrate reductase (niaD) mutants were isolated by selecting for 5-FOA and chlorate resistance,
respectively, with the precise genetic defect determined
using defined growth tests (see materials and methods). The pyrG and niaD genes were chosen due to
the availability of positive selection regimes that allow
spontaneous mutants to be isolated, the nonleaky phenotypes of the mutants under selection conditions, and
the availability of heterologous pyrG and niaD genes for
use as transformation markers. The mutations that gave
rise to the 5-FOA and chlorate resistance in SPM4 were
confirmed by complementation using the cloned A. nidulans pyrG (AnpyrG) and A. oryzae niaD (AoniaD) genes
(Oakley et al. 1987; Unkles et al. 1989).
A transformation procedure was developed based on
polyethylene glycol-mediated cell fusion using P. marneffei
protoplasts. Protoplasting trials showed that germlings
grown for 40 hr at 37⬚ produced cells with the greatest
protoplasting efficiency (data not shown). These growth
conditions were used for subsequent transformation experiments and were optimized for digestion time. DNAmediated transformation of the protoplasts used a technique developed for A. nidulans protoplasts that makes
use of polyethylene glycol-induced protoplast fusion to
introduce the transforming DNA into the cytosol (Tilburn et al. 1983; Yelton et al. 1984). This technique
was also shown to be effective in P. marneffei using the
same polyethylene glycol concentrations and times used
for A. nidulans transformations (Andrianopoulos and
Hynes 1988).
The pAB4342 (AnpyrG) and pSTA14 (AoniaD) plasmids were shown to complement the pyrG and niaD
mutant phenotypes of SPM4 at frequencies up to 1400
transformants per microgram of plasmid DNA. This efficiency was dependent on both the concentration of
DNA used and whether the DNA was circular or linear
(Table 2). Southern blot analysis of the pyrG⫹ transformants showed that pAB4342 DNA was present in the
genome of the transformants and was associated with
high molecular weight genomic DNA rather than being
present as independent episomes (Figure 3). Estimates
of plasmid copy number in the transformants ranged
from a single copy to multiple copies that were often
integrated in tandem. Analysis of transformants generated with linearized plasmid DNA showed that most
integration events were single copy, accounting for the
reduced transformation frequency observed when this
type of DNA was used.
The bleomycin resistance plasmid pAmPh520 (Austin et al. 1990) was also shown to be an effective dominant selectable marker. Transformants obtained using
this plasmid were isolated at a frequency lower than
that for the pSTA14 and pAB4342 plasmids with 150
transformants obtained per microgram of pAmPh520
(data not shown). Selection schemes based on other
dominant selectable markers such as hygromycin resis-
1008
A. R. Borneman, M. J. Hynes and A. Andrianopoulos
TABLE 2
Transformation efficiency of P. marneffei
Plasmid
Marker
gene
pAB4342
(pyrG)
pSTA14
(niaD)
DNA (ng)
No. of
transformants/107
protoplastsa
Transformation freq.
(transformants/␮g/107
protoplasts)
50
100
1,000
2,000
10,000
200b
50
100
1,000
2,000
70
138
110
120
390
25b
65
125
100
100
1,400
1,380
110
60
38
125b
1,300
1,250
100
50
a
The averages for at least two independent experiments are shown with original data averaged to the nearest
multiple of 10.
b
Transformation using linear pAB4342 DNA.
tance were shown to be unsuitable for use in P. marneffei
due to high-level natural resistance.
Cotransformation has previously been shown to be
extremely useful for transforming fungi with plasmids
that do not possess a selectable marker (Fincham 1989;
Hynes 1996). Cotransformation was tested in P. marneffei
by transforming SPM4 with a combination of pAB4342
and the plasmid pRS31, which encodes GFP fused to
the region encoding the N terminus of the A. nidulans
StuA protein expressed constitutively from the gpdA promoter. This protein fusion has previously been shown to
localize GFP to the nucleus of A. nidulans transformants
(Suelmann et al. 1997). A total of 50 pyrG⫹ transformants
were chosen at random and screened for GFP expression and localization. Of the 50 transformants tested,
46% displayed GFP fluorescence that was localized to
the nucleus in both the filamentous and yeast forms
(Figure 4). Southern analysis confirmed the presence
of both plasmids in the genomes of the GFP-expressing
transformants (data not shown).
Targeted gene deletion of the stlA locus: To examine
the role of stlA, a targeted deletion of the stlA locus was
obtained. Plasmid pAB4625 was created by inserting
the AnpyrG cassette of pAB4342 into pAB4624, thereby
deleting the majority of the stlA coding region including
the predicted translational start codon, homeodomain,
and both C2H2 fingers. Strain SPM4 was transformed
with pAB4625, previously linearized by restriction enzyme digestion at a unique ApaI site to increase the
frequency of homologous integration at the stlA locus
(Figure 5A). A total of eight pyrG⫹ transformants were
obtained from 500 ng of digested plasmid. None of the
transformants displayed any detectable phenotype when
grown at 25⬚. All eight transformants were screened by
Southern blot analysis and one of these transformants,
TAB19008, displayed a genomic restriction pattern consistent with deletion of the stlA locus and integration of a
single copy of the pyrG selectable marker (Figure 5B).
stlA is not required for growth or development: The
⌬stlA strain (TAB19008) was examined for any phenotypes that may be associated with loss of stlA function.
There were no detectable differences in growth rate,
colony morphology, or conidiation density observed at
25⬚ during filamentous growth. Microscopic examination showed that the ⌬stlA strain had normal hyphal and
conidiophore morphology (Figure 6A). At 37⬚, colony
morphology and growth rate were shown to be normal
and both the wild-type and ⌬stlA strains were capable
of switching from the yeast to filamentous growth forms
following transfer from 37⬚ to 25⬚ (Figure 6B). In addition, the ⌬stlA strain showed normal yeast cell morphology at the microscopic level, including normal positioning of nuclei (4⬘6-diamidino-2-phenylindole staining)
and septa (calcofluor staining; data not shown).
stlA can complement the sexual defect of an A. nidulans steA mutant: To assess whether the stlA gene actually
encoded a functional homolog of STE12, we tested the
ability of stlA to complement the sexual cycle defect of
the A. nidulans steA deletion strain UI139, which is selfsterile and is therefore unable to form cleistothecia unless crossed to an steA⫹ strain (Vallim et al. 2000).
UI139 was cotransformed with pAmPh520 (ble R; Austin et al. 1990) and pAB4624 (stlA). Bleomycin-resistant
transformants were picked at random and tested for
their ability to produce cleistothecia without outcrossing. From 50 colonies screened, 34 were shown to produce morphologically normal cleistothecia, with two
representative colonies shown in Figure 7. Cleistothecia
from several of these transformants were analyzed and
shown to contain viable ascospores. Southern blot analysis of these transformants showed that all the complemented strains contain copies of the stlA gene while
retaining the steA deletion locus (data not shown).
StlA is conserved at the DNA level: P. marneffei stlA
encodes a functional gene product capable of activating
the sexual developmental pathway in an A. nidulans steA
An STE12 Homolog from P. marneffei
1009
ment in both of these organisms (Borneman et al. 2000),
show similar values of 0.091 for the entire coding region
and 0.004 for the ATTS DNA-binding motif. Similarly,
comparison of the conserved region from homologous
chitin synthase genes in these two organisms, P. marneffei
chsA (PmCHS1, accession no. U60515) and A. nidulans
chsC (accession no. AB023911), yielded a dN/dS rate of
0.0261.
DISCUSSION
Figure 3.—Analysis of the fate of plasmid DNA in P. marneffei
transformants. (A) Southern hybridization of genomic DNA
from 18 independent transformants using differing amounts
and forms of DNA (as indicated) digested with PstI, transferred
to membrane, and probed with a 1.4-kb, 32P-labeled PstI-BamHI
fragment of pAB4342 containing the A. nidulans pyrG gene.
PstI does not cut within pAB4342, therefore producing a single
hybridizing band per integrated plasmid copy. (B) Southern
hybridization of undigested genomic DNA from the identical
transformants used in A and probed using the same fragment
of pAB4342. Hybridizing sequences are associated with highmolecular-weight DNA, suggesting a chromosomally integrated location.
mutant. If stlA plays an important role in P. marneffei,
then it should still be under selective pressure. To assess
this, a comparison of the synonymous and nonsynonymous base substitution rates (dN/dS) was performed on
the entire coding region of the P. marneffei and A. nidulans genes, as well as the two putative DNA-binding
motifs, which show the highest level of conservation.
Synonymous and nonsynonymous nucleotide substitutions were assigned, counted, and corrected for multiple
substitutions on a codon-for-codon basis of DNA alignments. The dN/dS rates were 0.069, 0.011, and 0.010
for the entire coding region, the homeodomain DNAbinding motif, and the C2H2 DNA-binding motif, respectively. The P. marneffei and A. nidulans abaA genes, which
have been shown to be required for asexual develop-
P. marneffei as a model for fungal dimorphism: P. marneffei is a pathogen that is rapidly emerging as a public
health problem, not only in Asia where P. marneffei has
been classed as an “AIDS defining pathogen,” but also
throughout other parts of the world with cases of P. marneffei infection having been recorded in Australia, Europe, and North America (Cooper 1998; Cooper and
Haycocks 2000). Like a number of other fungal pathogens, P. marneffei displays a temperature-dependent dimorphic switch; however, unlike most other dimorphic
fungi that are classified as yeasts, P. marneffei is primarily
a filamentous species that adopts a yeast morphology
in its host. The control of this developmental process
is of interest because, with other dimorphic fungi, it
is thought that the dimorphic switch is required for
pathogenicity and may therefore provide a target for
controlling infection. To investigate these processes in
P. marneffei at the molecular level, we have isolated mutant strains and developed a protocol to transform them.
We have also demonstrated that it is possible to create
precise mutations in genes by targeted gene replacement. These techniques provide the basic tools required
for in-depth study of P. marneffei pathogenicity and development at the genetic level through the use of gene
knockout strains and gene reporter and protein localization constructs.
P. marneffei has a conserved STE12 homolog that is
distinct from S. cerevisiae STE12: P. marneffei stlA was
shown to be highly conserved when compared to the
recently cloned A. nidulans homolog, steA (Vallim et al.
2000). The predicted StlA protein, like A. nidulans SteA
and C. neoformans Ste12␣, possesses two potential DNAbinding domains, a homeodomain found in all Ste12p
homologs, in addition to two C2H2 Zn2⫹ motifs. These
three proteins clearly define a second class of Ste12
protein, present in higher ascomycetes and basidiomycetes, which is distinct from the yeast proteins that lack
the C2H2 Zn2⫹ motif. The P. marneffei, A. nidulans, and
C. neoformans proteins also lack homology to the regions
that are important for the regulation of Ste12p by Dig1p
and Dig2p (Pi et al. 1997). These proteins may therefore
be regulated directly by a MAPK without the requirement for Dig1p or Dig2p homologs or, alternatively,
the C2H2 Zn2⫹ finger domains may be responsible for
regulating the activity of the proteins through proteinprotein interactions (Mackay and Crossley 1998).
StlA is a conserved gene: STE12 homologs in other
1010
A. R. Borneman, M. J. Hynes and A. Andrianopoulos
Figure 4.—Microscopic analysis of pAB4342-pRS31 cotransformants expressing GFP. A representative cotransformant for the
StuA-GFP-expressing pRS31 plasmid is shown. Colonies were grown on microscope slides covered with a thin layer of SD solid
medium and incubated at either 25⬚ for 2 days to induce filamentous growth (A) or 37⬚ for 7 days to induce yeast growth (B).
Slides were examined using both differential interference (DIC) and fluorescence optics (GFP) to detect the cellular localization
of the StuA-GFP protein. Both the filamentous and yeast-like growth forms show the StuA-GFP protein localized in the nucleus,
as noted by the concentrated fluorescence in these organelles.
fungi are required for sexual development, yeast-hyphal
switching, or both. Although deletion of the stlA locus
from P. marneffei has no discernible effect on growth,
development, or dimorphic switching, there are a number of lines of evidence that suggest stlA plays a role in
P. marneffei. First, the P. marneffei gene shows a highly
regulated pattern of expression with relatively high levels in vegetative hyphal cells and yeast cells. Second, the
stlA gene is able to complement the sexual development
defect of an A. nidulans steA mutant, showing that the
P. marneffei stlA gene is correctly regulated at the tran-
scriptional level and that the protein is fully functional
in A. nidulans. Third, measurements of the rate of synonymous and nonsynonymous base substitutions in the
stlA and steA genes show that there is a strong bias against
nonsynonymous changes over the entire coding region
but especially for the two putative DNA-binding motifs.
In comparison to the stlA/steA genes, the abaA developmental regulatory genes, which are required for asexual
development and yeast cell morphogenesis in P. marneffei
and for asexual development in A. nidulans (Boylan
et al. 1987; Andrianopoulos and Timberlake 1994;
An STE12 Homolog from P. marneffei
1011
Figure 5.—Deletion of the stlA by targeted
gene replacement. (A) Restriction maps of the
wild-type (top) and predicted gene-replaced stlA
(bottom) loci showing the position of the stlA
coding region (black boxes) and the region replaced by the A. nidulans pyrG selectable marker
(shaded box). The region of stlA DNA used as a
probe for the Southern blotting analysis is represented by an open rectangle. (B) Southern blot
analysis of the wild type and eight transformants
obtained from the ApaI digested pAB4625 deletion construct indicated in A. Genomic DNA was
isolated from each transformant (labeled 1–8)
and the recipient strain (W), restriction digested
(Sal I or Bgl II), gel fractionated, and Southern
blotted. The Southern blot was probed using the
P32-labeled stlA fragment indicated in A. The stlA
deletion transformant, TAB19008, which has the
AnpyrG marker integrated at stlA, is indicated by
an asterisk.
Borneman et al. 2000), show a slightly weaker bias
against nonsynonymous changes over the entire coding
region but a stronger bias over the DNA-binding domain. This suggests that very similar selective pressures
are operating for both pairs of genes. Similar results
were obtained for a pair of chitin synthase homologs.
P. marneffei may have a sexual cycle: Although the
coding regions of stlA and steA are highly conserved,
the promoter regions share very little similarity and
searches for binding sites of known transcription factors
failed to identify any motifs conserved between species.
This result, coupled with the different transcriptional
profiles of the P. marneffei and A. nidulans genes, demonstrates the divergence between these two fungi. There-
Figure 6.—Loss of stlA does not affect vegetative growth or conidiation in P. marneffei. (A)
Wild-type (FRR2161) and ⌬stlA (TAB19008)
strains were grown on ANM ⫹ GABA medium
at 25⬚ for 10 days to examine hyphal growth
and conidiation. The edges of single colonies
show the fluffy appearance indicative of conidiophores (left). Individual conidiophores were
also examined by growing both wild type and
⌬stlA on slides coated in ANM ⫹ GABA for 5
days. Colonies were fixed and viewed using
DIC microscopy to examine the structure of
individual conidiophores (right). Bar, 20 ␮m.
(B) Wild-type (FRR2161) and ⌬stlA (TAB19008) strains were grown on SD media for 4
days at 37⬚ to examine yeast growth. Single
colonies were viewed using brightfield microscopy (left). The ability to undertake the yeasthyphal transition was then investigated by shifting the plates to 25⬚ for 3 days to induce the
dimorphic switch and result in hyphal growth
(right).
1012
A. R. Borneman, M. J. Hynes and A. Andrianopoulos
Figure 7.—stlA can complement the ⌬steA
mutation in A. nidulans. The sexual cycle was
induced in wild type (steA⫹), UI139 (⌬steA),
and UI139 transformed with stlA by incubation
at 37⬚ for 14 days. Wild-type strains of A. nidulans form numerous cleistothecia (white arrowheads). UI139 does not produce cleistothecia
unless mated to an steA⫹ strain. UI139 was cotransformed with pAB4624 (stlA) and pAmPh520 (ble R). Transformants that had regained
the ability to form cleistothecia without outcrossing (white arrowheads) were isolated, indicating complementation of the steA phenotype. Cleistothecia formed by two
representative transformants (TAB770002 and TAB770017) are shown.
fore, the high degree of conservation of stlA in P. marneffei
may be due to an involvement in controlling sexual
development and to the fact that P. marneffei has a previously undefined teleomorphic (sexual) state. This is
supported by the identification of an A. nidulans stuA
homolog (which is also involved in controlling mating)
in P. marneffei, which, like stlA, is highly conserved between the two species (Miller et al. 1992; A. R. Borneman, M. J. Hynes and A. Andrianopoulos, unpublished
results). This may be a similar situation to C. albicans,
which was also thought to be strictly asexual, but from
which numerous, highly conserved homologs of S. cerevisiae mating genes had been identified (Sadhu et al.
1992; Leberer et al. 1996; Raymond et al. 1998). Recently, it has been shown that C. albicans diploids possess
homologs of both the a and ␣ mating loci of S. cerevisiae.
Deletion of one of each of these mating loci from reciprocal strains allows C. albicans to be induced to mate,
albeit in an artificial manner, to form tetraploid cells
(Hull et al. 2000; Magee and Magee 2000).
Alternatively, loss of sexual reproduction in P. marneffei
may be a relatively recent event. Phylogenetic studies
of teleomorphic Talaromyces species with Penicillium
asexual states and anamorphic (asexual) Penicillium
species of the Biverticilliate group have clearly demonstrated that sexual and asexual species are closely related. The asexual species also appear to be relatively
recently derived, probably due to loss of the sexual cycle
(LoBuglio et al. 1993). P. marneffei is a member of the
biverticilliate group and has a number of closely related
teleomorphic species (LoBuglio and Taylor 1995).
Studies of members of the Aspergilli also show close
phylogenetic pairing between a teleomorphic and an
anamorphic species (Geiser et al. 1996).
If P. marneffei is capable of sexual reproduction, there
are a number of reasons why the sexual state may not
have been observed. P. marneffei may be heterothallic,
requiring two strains of different mating type to be
brought together for mating to be successful. If one
mating type of P. marneffei is rare, avirulent, or even
monomorphic, it may not have been isolated or may
have failed to be identified as P. marneffei. While this
seems unlikely, a similar situation exists in C. neoformans
Serotype D strains where the MAT␣ strain is 30-fold more
prevalent in the environment and 40-fold more prevalent in infections than the MATa strain (Kwon-Chung
and Bennett 1978). The MAT␣ strain has also been
shown to be more virulent than the MATa strain (KwonChung et al. 1992). This dichotomy is more pronounced
in Serotype A isolates of C. neoformans as a MATa strain
of this serotype has never been isolated. If Serotype A
were considered in isolation from Serotype D, it would
be classed as asexual due to the lack of an opposite
mating type. This situation may also be complicated
further by environmental constraints on P. marneffei mating. There may be any number of stimuli that may be
required for mating between strains and may include
infection of multiple strains in one host, as was the case
observed for the artificially induced mating of C. albicans
(Hull et al. 2000). Further work is therefore required
in this area to discriminate among the many possibilities
that are presented with respect to the sexuality of P.
marneffei and the function of stlA. The presence of an
STE12 homolog in an “asexual” species, however, makes
this gene an excellent marker for the study of the evolution of sexual/asexual life cycles in fungi.
We thank Dave Geiser and Jon Martin for their helpful discussions
and Rhonni Croft for her expert technical assistance. This work was
supported by an Australian Research Council grant. A.R.B. was supported by an Australian Postgraduate Award (APA) scholarship.
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