A Large-Scale Complex Haploinsufficiency-Based Genetic Interaction Screen in Candida albicans: Analysis of the RAM Network during Morphogenesis
et al. (2011) A Large-Scale Complex Haploinsufficiency-Based Genetic Interaction Screen
in Candida albicans: Analysis of the RAM Network during Morphogenesis. PLoS Genet 7(4): e1002058. doi:10.1371/journal.pgen.1002058
A Large-Scale Complex Haploinsufficiency-Based Genetic Interaction Screen in Candida albicans : Analysis of the RAM Network during Morphogenesis
Nike Bharucha 0
Yeissa Chabrier-Rosello 0
Tao Xu 0
Cole Johnson 0
Sarah Sobczynski 0
Qingxuan Song 0
Craig J. Dobry 0
Matthew J. Eckwahl 0
Christopher P. Anderson 0
Andrew J. Benjamin 0
Damian J. Krysan 0
Michael Snyder, Stanford University School of Medicine, United States of America
0 1 Department of Molecular, Cellular, and Developmental Biology, University of Michigan , Ann Arbor , Michigan, United States of America, 2 Department of Pediatrics, University of Rochester School of Medicine and Dentistry , Rochester , New York, United States of America, 3 Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry , Rochester, New York , United States of America
The morphogenetic transition between yeast and filamentous forms of the human fungal pathogen Candida albicans is regulated by a variety of signaling pathways. How these pathways interact to orchestrate morphogenesis, however, has not been as well characterized. To address this question and to identify genes that interact with the Regulation of Ace2 and Morphogenesis (RAM) pathway during filamentation, we report the first large-scale genetic interaction screen in C. albicans. Our strategy for this screen was based on the concept of complex haploinsufficiency (CHI). A heterozygous mutant of CBK1 (cbk1D/CBK1), a key RAM pathway protein kinase, was subjected to transposon-mediated, insertional mutagenesis. The resulting double heterozygous mutants (6,528 independent strains) were screened for decreased filamentation on Spider Medium (SM). From the 441 mutants showing altered filamentation, 139 transposon insertion sites were sequenced, yielding 41 unique CBK1-interacting genes. This gene set was enriched in transcriptional targets of Ace2 and, strikingly, the cAMP-dependent protein kinase A (PKA) pathway, suggesting an interaction between these two pathways. Further analysis indicates that the RAM and PKA pathways co-regulate a common set of genes during morphogenesis and that hyperactivation of the PKA pathway may compensate for loss of RAM pathway function. Our data also indicate that the PKAregulated transcription factor Efg1 primarily localizes to yeast phase cells while the RAM-pathway regulated transcription factor Ace2 localizes to daughter nuclei of filamentous cells, suggesting that Efg1 and Ace2 regulate a common set of genes at separate stages of morphogenesis. Taken together, our observations indicate that CHI-based screening is a useful approach to genetic interaction analysis in C. albicans and support a model in which these two pathways regulate a common set of genes at different stages of filamentation.
Funding: This work was supported by National Institute of Allergy and Infectious Diseases grants 5R21AI084539 (to DJK and AK) and 5T32AI007464 (to YC-R), as
well as by an American Cancer Society grant RSG-06-179-01-MBC (to AK). The funders had no role in study design, data collection and analysis, and decision to
publish or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Candida albicans is a member of the resident flora of the
gastrointestinal tract and is the most common fungal pathogen in
humans. The most severe manifestations of candidiasis occur in
immunocompromised patients and include debilitating mucosal
disease such as oropharyngeal candidiasis as well as life-threa
tening disseminated infections of the bloodstream and major organ
systems . Animal studies have shown that the pathogenic
potential of C. albicans is associated with its ability to transition
between three morphological states: yeast, pseudohyphae, and
hyphae [2,3]. Further insights into the contributions of the
different morphotypes to pathogenesis have emerged from elegant
studies with C. albicans strains that allow the conditional induction
of filamentation in vivo . For example, C. albicans genetically
restricted to the yeast form by constitutive expression of NRG1 are
able to establish infection in mice but no disease results until the
expression of NRG1 is repressed and the organism is able to form
The relationship between morphogenesis and virulence in C.
albicans is, however, not a simple one. Many mutants that are
unable to undergo morphogenesis also display other phenotypes.
For example, many transcription factors that are required for
morphogenesis regulate a host of other genes and display
pleiomorphic phenotypes. The complicated nature of the relationship
between morphogenesis has been further highlighted by the
elegant study recently reported by Noble et al. . Noble et al.
generated a bar-coded collection of homozygous deletion mutants
and used it in a signature-tagged mutagenesis study of infectivity in
a mouse model . Mutants with defects in morphogenesis were
Candida albicans is the most common cause of fungal
infections in humans. As a diploid yeast without a classical
sexual cycle, many genetic approaches developed for
large-scale genetic interaction studies in the model yeast
Saccharomyces cerevisiae cannot be applied to C. albicans.
Genetic interaction studies have proven to be powerful
genetic tools for the analysis of complex biological
processes. Here, we demonstrate that libraries of C.
albicans strains containing heterozygous mutations in
two different genes can be generated and used to study
genetic interactions in C. albicans on a large scale. Double
heterozygous mutants that show more severe phenotypes
than strains with single heterozygous mutations are
indicative of genetic interactions through a phenomenon
referred to as complex haploinsufficiency (CHI). We applied
this approach to the study of the RAM (Regulation of Ace2
and Morphogenesis) signaling network during the
morphogenetic transition of C. albicans from yeast to
filamentous growth. Among the genes that interacted
with CBK1, the key signaling kinase of the RAM pathway,
were transcriptional targets of the RAM pathway and the
protein kinase A pathway. Further analysis supports a
model in which these two pathways co-regulate a
common set of genes at different stages of filamentation.
more likely to have decreased infectivity; however, a significant
portion of mutants with severe morphogenesis defects retained the
ability to cause infection. It is important to note that Noble et al.
assayed for infection and not for disease. Thus, their results are not
necessarily in conflict with studies discussed above that indicate
that morphogenesis is required for disease progression in animal
models . Furthermore, their work serves to highlight the fact
that additional studies will be required to fully understand the
complex relationship between morphogenesis and pathogenesis in
Given the close association of morphogenesis with C. albicans
pathogenesis, the genetic and cell biologic analysis of this process
has been the subject of intensive study . Consequently, many
genes have been shown to affect filamentation, and,
correspondingly, a number of regulatory pathways have been shown to play a
role in the orchestration of the morphogenetic program in C.
albicans . The PKA, CPH1, HOG1, RIM101, CHK1, and CBK1/
RAM pathways are among those that regulate morphogenesis
under a variety of conditions [6,7]. Although much remains to be
learned about how individual pathways and genes contribute to
morphogenesis, an important question that has not been
extensively studied is how these various pathways interact to
In the model yeast S. cerevisiae, relationships between regulatory
pathways can be readily characterized using recently developed
systematic, genome-wide genetic interaction strategies .
These approaches have yielded a wealth of information regarding
the mechanisms through which cells regulate complex biological
processes . However, because C. albicans is diploid and lacks a
classical meiotic cycle, the mating-based genetic strategies used to
create genome-wide libraries of double mutant strains in S.
cerevisiae are not applicable. Consequently, genetic interaction
studies in C. albicans have been limited to gene-by-gene analyses.
Despite these limitations, such studies have proven quite
informative and suggest that large scale interaction studies could
represent a powerful approach to studying regulatory networks in
C. albicans. For example, Braun et al. carried out a thorough,
systematic epistasis analysis of three transcriptional regulators
(EFG1, TUP1 and CPH1) and showed that each played a distinct
role in the regulation of filamentation .
Recent advances in the genetic analysis of C. albicans have
greatly facilitated the development of innovative approaches to the
study of this important human pathogen . Among these
important developments is the application of transposon-based
mutagenesis strategies  to the creation and study of large-scale
libraries of heterozygous [15,16] and homozygous [17,18] C.
albicans mutants. Similarly, large collections of homozygous null
 and conditional mutants  have been created in a targeted
manner and analyzed for a variety of phenotypes including
morphogenesis, virulence and drug susceptibility. To our
knowledge, one area that has not been explored is the development of
approaches to large-scale synthetic genetic interaction analysis in
Here, we describe the first large-scale synthetic genetic
interaction screen in C. albicans. Our strategy builds on pioneering yeast
genetics approaches developed in both S. cerevisiae and C. albicans
and is based on the concept of complex haploinsufficiency (CHI).
CHI is a special case of a genetic phenomenon referred to as
unlinked non-complementation in the context of yeast genetics
and as dominant enhancers or dominant modifiers when applied
to Drosophila . Unlinked non-complementation occurs when a
cross between two haploid strains containing single recessive
mutations located in separate loci results in a diploid strain
(complex heterozygote) that retains the phenotype of a parental
strain. In yeast, the construction of such mutants was used to great
advantage in the genetic analysis of cytoskeletal genes such as
tubulin  and actin . CHI, which is a special case of
unlinked non-complementation, occurs when strains containing
heterozygous mutations at two separate loci display a more severe
phenotype than strains that contain heterozygous mutations at the
single loci alone . In essence, CHI can also be called synthetic
haploinsufficiency. Recently, a genome-wide CHI-based strategy
was developed in S. cerevisiae and successfully used to create a
genetic interaction network for the essential gene, ACT1 .
As described in the seminal work of Uhl et al. , large scale
haploinsufficiency-based screening was first applied to C. albicans in
the transposon-mediated, insertional mutagenesis analysis of
filamentation and, thus, haploinsufficiency-based screening has
excellent precedence in this system. Whereas Uhl et al. carried out
their haploinsufficiency screen starting with a wild type strain
, we reasoned that transposon mutagenesis of a parental strain
containing a heterozygous mutation at a locus of interest would
represent an expedient approach to the generation of a large
library of complex heterozygotes that could then be the basis of a
CHI screen for genes that interact with the parental mutant.
In principle, CHI-based screening has a number of attractive
features. First, CHI allows one to identify genes that function
within the pathway affected by the parental or query mutation
including upstream and downstream components of the
pathway, transcriptional outputs of the pathway, and substrates
of pathway enzymes. Second, CHI-based screening can also
identify genes or pathways that function in parallel with the
query pathway and, therefore, allow one to identify pathways
that co-regulate a given process. Third, CHI is ideal for the
study of essential genes because only heterozygous mutations are
We developed a CHI-based screening strategy (Figure 1) and
applied it to the identification of genes that interact with the
RAM signaling network during C. albicans filamentation [24
27]. The RAM network has been extensively studied in S.
cerevisiae  and is required for a variety of cellular processes
in both S. cerevisiae and C. albicans including polarity, cell wall
synthesis, cell separation and filamentous growth. Cbk1 is the
key serine/threonine protein kinase [24,27] that mediates
many of the functions of the RAM network through its
regulation of the transcription factor Ace2 [24,27]. RAM
pathway mutants in C. albicans show two distinct filamentation
phenotypes: CBK1 null mutants are unable to form filaments
on Spider Medium (SM) or serum-containing medium [24,27]
while ACE2 null mutants are constitutively pseudohyphal and
form true hyphae on serum . Although our understanding
of the RAM network in C. albicans has increased in recent years
, many questions remain, including: how does it
interact with the many other regulatory pathways during
morphogenesis and what genes and proteins are regulated by
Cbk1 and/or its downstream transcription factor Ace2?
Through this novel application of a CHI-based screening
strategy, we have identified RAM/Ace2 transcriptional targets
and generated genetic evidence for an interaction between the
RAM and PKA pathways during morphogenesis. Follow-up
studies of the screening results further suggest that a balance
between RAM and PKA-pathway activity is required for cells
to establish a normal distribution of morphotypes during
nutrient-induced filamentation. Taken together with previous
work on these two pathways, our observations support a model
where PKA-regulated transcriptional activity is most important
in the transcription of RAM/PKA co-regulated genes early in
morphogenesis, while the RAM pathway is more important as
daughter nuclei accumulate within the hyphal structure.
CHI screening of the cbk1D/CBK1 mutant
The cbk1D/CBK1 mutant was originally studied in C. albicans by
McNemar and Fonzi  and was found to be haploinsufficient
with respect to filamentation on a variety of media. Uhl et al. also
isolated a heterozygous cbk1 insertion mutant in their
haploinsufficiency screen . As shown in Figure 2A, cbk1D/CBK1 colonies
show a decreased area of central wrinkling and a more prominent
ring of peripheral filamentation on SM at 37uC. The
haploinsufficiency of this parental strain was advantageous for two reasons.
First, it provided increased sensitivity in that the strain was already
deficient for filamentation. Second, it could also improve
specificity because weak phenotypes of non-interacting,
transposon-derived mutants would not be apparent due to masking by the
As described above [24,25,27], RAM pathway mutants show
two distinct phenotypes depending on the conditions used to
induce filamentation, but both phenotypes are apparent on solid
Spider Medium (SM). In order to identify mutations that
potentially interacted with both general functions of the pathway,
we, therefore, screened for decreased filamentation on SM at
37uC. All subsequent experiments were conducted under these
conditions unless otherwise indicated.
The library of 6528 complex heterozygous mutants was spotted
in 96-well format and scored for decreased peripheral invasion and
altered colony wrinkling relative to a Ura+ derivative of the
parental cbk1D/CBK1 strain (11, Figure 2A). Clones showing both
phenotypes were re-tested using two independent colonies. A total
of 441 complex heterozygous mutants with decreased peripheral
invasion and altered colony wrinkling were re-confirmed on both
SM and SM containing uridine to control for positional effects of
the URA3 gene (Figure 2A). We specifically selected mutants with
decreased zones of peripheral filamentation and less pronounced
central wrinkling relative to the parental strain (Figure 2A and 2B).
All mutants showed some degree of peripheral filamentation. The
most common composite phenotype indicated a small zone of
peripheral agar invasion with a broad region of moderate
wrinkling (Figure 2B).
The transposon insertion sites for approximately one-third of
the mutants (139 strains) showing potential synthetic genetic
interactions were identified using a 39-RACE/sequencing
approach (see Materials and Methods), yielding 42 unique
transposonderived mutations as putative CBK1-interactors. Since 8 insertion
sites were identified in at least 5 separate clones (Figure 3A and
3B), the screen appeared to be saturated to the limits of the library
and the mutagenesis technique. Therefore, we did not sequence
the remaining two-thirds of the mutants and focused on evaluating
the initial set of 42 mutants. It is, however, important to note that
the screen itself is unlikely to be saturated for all possible CBK1
interactors, as the library almost certainly did not contain
insertions in all predicted C. albicans genes.
The URA3 marker was recycled from the heterozygotes by
5FOA mediated recombinational excision . Following
phenotypic re-testing to confirm that homozygosis was not responsible for
curing the URA3 marker, CBK1 was re-integrated at its
chromosomal position using plasmid pMM4 . The phenotypes of 41 of
42 candidate CHI strains were modified by re-integration of CBK1
(Figure 2A), indicating that the observed phenotypes were
dependent on the cbk1 mutation and were likely due to a synthetic genetic
interaction between cbk1D/CBK1 and the transposon insertion. The
high percentage of CBK1-dependent phenotypes may be due to the
fact that the parental cbk1D heterozygote is itself haploinsufficient on
SM and most non-interacting insertion mutations that are
themselves haploinsufficient do not have sufficiently strong phenotypes to
appreciably change the phenotype of the double heterozygote
relative to the parental strain. To confirm these interactions further,
a subset of ten complex heterozygous mutants was independently
constructed from CAMM-292 by single gene-replacement . All
ten double mutants recapitulated phenotypes displayed by the
transposon-derived mutants and showed distinct phenotypes
relative to strains with single deletions of the interacting genes.
Representative images from this analysis are shown in Figure 2B.
To further characterize the morphologies of the mutants, we
determined the proportion of yeast, pseudohyphae and hyphae
after 3 hours induction in liquid SM at 37uC. The interacting
mutants consistently showed an increased proportion of
pseudohyphal cells relative to wild type and cbk1D/CBK1 strains
(Figure 2C). Similarly, examination of cells scraped from SM
plates showed that the filaments of double mutants had constricted
septal areas characteristic of pseudohyphae (Figure 2D).
Importantly, all of the mutants were indistinguishable from wild type and
the parental strain when serum was used as the inducer of
filamentation (data not shown). Since ace2D/D mutants also show
decreased peripheral invasion, decreased central wrinkling,
increased levels of pseudohyphae, and normal filamentation on
serum (25), we conclude that the majority of the CBK1-interacting
genes isolated in the screen appear to affect the Ace2-dependent
functions of the RAM pathway.
The set of CBK1-interactors contains genes related to
Literature analysis of the set of CBK1-interactoring genes revealed
that approximately one-half are involved in glycolysis/respiration,
biosynthesis, or cell wall metabolism (Figure 3B), cell processes
Figure 3. Summary and bioinformatic analysis of screening data. (A) Summary of screening results and list of CBK1-interacting genes. (B) List
of CBK1-synthetic genetic interactions during morphogenesis grouped according to three most common GO terms. Colors indicate the number of
times each insertion was isolated. (C) Venn diagram depicting the number of genes putatively co-regulated by the RAM and PKA pathways. (D) List of
CBK1-interacting genes with Ace2 and both Ace2/Efg1 consensus binding sites within the region 1000 bp upstream of the start codon.
consistent with established functions of the RAM pathway .
An important interactor in terms of validating the screen is SSD1
because it is a likely Cbk1 substrate in S. cerevisiae , displaying
well-characterized genetic interactions with CBK1 in both S. cerevisiae
 and C. albicans . Comparison of our dataset with that
generated by the haploinsufficiency screen of Uhl et al. revealed no
overlap . As discussed above, we suspect that this lack of overlap
is also related to the fact that our parental strain is haploinsufficient
for filamentation and, thus, non-interacting transposon-derived
mutations causing simple haploinsufficiency were, in effect, masked
by the phenotype of the parental strain.
In principle, the Ace2-deficient phenotypes displayed by the
double heterozygous mutants could result from mutations that
interfere with the activation of Ace2 or from mutations that affect
a key transcriptional target of Ace2. We isolated three mutants
that could cause a CHI-interaction with CBK1 through the former
mechanism. First, we isolated orthologs of two genes that regulate
mitotic exit in S. cerevisiae, CDH1  and SLK19 . Ace2 is well
known to localize to the nuclei of daughter cells in both S. cerevisiae
 and C. albicans [25,39]. Since Cdh1 and Slk19 regulate mitotic
exit, the point in the cell cycle when Ace2 localizes to the nuclei
, we suggest that disruption of mitotic exit through the loss of
these proteins may further decrease the overall activity of Ace2. In
addition, NSP1, a key component of the nuclear import
machinery, was isolated. Studies in S. cerevisiae  have indicated
that decreased NSP1 gene dosage leads to inhibition of nuclear
import, and it seems plausible that a strain lacking an allele of
NSP1 could have decreased nuclear import of Ace2 which would
further decrease the overall Ace2-mediated transcriptional activity
of the cbk1D/CBK1 mutant.
The larger class of CBK1-interacting mutants that relate to Ace2
function is the set of genes that appear to be part of the
transcriptional output of the RAM pathway (Figure 3C and 3D). To
identify such genes in our data set, we searched the promoter
regions of CBK1-interactors and found 22 genes that contain a C.
albicans Ace2-consensus binding sequence [MMCCASC, 26]. Of
these genes, 11 have been shown to display decreased expression
in ace2D/D mutants during hyphal induction as reported in a
recent transcriptional profiling study . To further confirm that
our screen identified genes regulated by Ace2, we examined the
binding of Ace2 to the promoters of 5 CBK1-interactors with
consensus binding sites (ACT1, ADH1, ENO1, HGT6, & RGD3)
during both yeast and hypha-phase growth by chromatin
immunoprecipitation (ChIP). Consistent with ChIP data for
Ace2 reported by Wang et al. , the absolute enrichment was
relatively low, most likely due to its cell cycle regulation and our
non-synchronous experiments (Figure 4). Nevertheless, all five
promoters were bound by Ace2 at levels comparable to those
observed for the well-established Ace2 target CHT3 and to those
reported by Wang et al.  during yeast growth. In addition,
three promoters were bound in hyphal phase (Figure 4). Taken
together, the presence of Ace2 binding sites, the transcriptional
profiling data, and ChIP data support the notion that many of the
CBK1-interacting genes are transcriptional targets of Ace2.
The CHI screen of cbk1D/CBK1 reveals potential
interaction between the RAM and PKA pathways during
Comparison of the set of CBK1-interactors with data from a
variety of transcriptional profiles of C. albicans morphogenesis
indicated that a substantial subset of CBK1-interactors (14
interactors, 34%) are regulated by the cAMP/PKA pathway
through the transcription factor Efg1 . Indeed, 10
CBK1interactors contain consensus binding sites for both Ace2 and Efg1
(Figure 3C and 3D), suggesting that these two transcription factors
may regulate a common set of genes. Further supporting this
notion are previous studies indicating that both Ace2 and Efg1
induce glycolytic genes and repress genes involved in oxidative
respiration [26,41]. Indeed, we searched the C. albicans genome
and found that the promoters of 384 genes contain consensus
binding sites for both Ace2 and Efg1 (Table S2). Consistent with
previous studies of the two pathways, the set of putatively
coregulated genes is enriched for genes contributing to glycolysis,
biosynthesis, and cellular stress responses. Recently, Wang et al.
have also shown that the promoters of Ace2-regulated cell wall and
cell separation genes are bound by both Efg1 and Ace2 during
morphogenesis . Taken together our genetic data strongly
support the notion that genes regulated by the PKA pathway may
also be important components of the transcriptional output of the
RAM pathway during morphogenesis.
In addition to transcriptional targets of the PKA pathway,
three other CBK1 interactors (MAF1, SLF1 & ACT1) have
connections to the PKA pathway (Figure 3A). MAF1 and SFL1
are both orthologs of PKA-regulated transcriptional regulators in
S. cerevisiae [42,43], suggesting that proper PKA-mediated
transcriptional control is important in the absence of full RAM
pathway activity. Further suggesting that the activity of the PKA
pathway is important in RAM pathway mutants, we isolated
ACT1 as a CBK1-interactor. Although ACT1 is, of course, a
crucial part of the cell cytoskeleton, it also plays an important role
in activation of the cAMP/PKA pathway. The Sundstrom lab
has shown that actin dynamics regulate PKA activity  and,
recently, Zou et al. have elegantly demonstrated that actin
functions as part of a PKA sensor/activator complex during
hyphal development . Indeed, decreased G-actin levels lead
to decreased PKA pathway activity and, in turn, decreased
filamentation in C. albicans . As such, one explanation for the
interaction between ACT1 and CBK1 is that the lowered ACT1
gene dosage in the act1D/ACT1 cbk1D/CBK1 mutant exacerbates
the filamentation defects of decreased RAM pathway activity by
concomitantly limiting PKA activity. This explanation also
implies that the PKA pathway may compensate for decreased
RAM pathway activity during morphogenesis.
Figure 4. The set of CBK1-interacting genes includes transcriptional targets of Ace2. The binding of Ace2-TAP to the promoter regions of 5
CBK1-interacting genes was assessed by ChIP in yeast and hyphae-phase cells (SM, 3 h, 37uC) containing a TAP-tag fused to the C-terminus of Ace2.
Bars indicate the ratio of promoter DNA (determined by PCR) in tagged extracts relative to un-tagged extracts (error bars indicate SD of three
replicates). Grey bars show promoters with increased abundance in tagged extracts, suggesting they are bound by Ace2. Pcht3, a known target of
Ace2 [13,21], and primers to a coding sequence (ORF cds) serve as positive and negative controls. A persistent contaminating band prevented
accurate assessment of ACT1 in hyphae.
RAM pathway mutants show evidence of increased PKA
To test the hypothesis that the RAM and PKA pathways
regulate a common set of genes during morphogenesis, we
examined the expression of two CBK1-interacting genes containing
both Ace2 and Efg1 binding sites in ace2D/D and efg1D/D mutants
after 3 hours of hyphal induction with SM. As shown in Figure 5A,
the expression of the transcripts increased in both strains relative
to wild type by quantitative RT-PCR. These observations suggest
either that Efg1 and Ace2 are functioning as transcriptional
repressors or that compensatory responses are occurring to
maintain expression of these genes during morphogenesis when
one of the two pathways is disabled.
To test the latter hypothesis, total cell lysates of the RAM
pathway mutant ace2D/D were prepared and the level of PKA
enzymatic activity determined after 3 hours exposure to
hyphainducing conditions (Figure 5B). At this time point, PKA activity
has reduced to low levels in wild type cells , but there is
clearly increased PKA activity in the ace2D/D mutant. This
suggests that the PKA pathway is hyperactive in RAM pathway
mutants and is consistent with the hypothesis that the PKA
pathway may compensate for decreased RAM pathway activity.
To further test the interaction between the RAM and PKA
pathways, we deleted one allele of CBK1 in strains containing
homozygous null mutations in one of the catalytic subunits of the
PKA enzyme  to yield the mutants cbk1D/CBK1 tpk1D/D and
cbk1D/CBK1 tpk2D/D. The two triple mutants along with wild
type and the parental mutants were incubated in SM for 3 hours
at 37uC to induce filamentation. As shown in Figure 5C, deletion
of TPK1 in the cbk1D/CBK1 background decreases the proportion
of pseudohyphae formed by the cbk1D/CBK1 mutant, while
deletion of TPK2 has no effect (data not shown), suggesting that
the increased proportion of pseudohyphae formed by cbk1D/
CBK1 is dependent on TPK1. The phenotypic differences evident
upon deleting the two isoforms of PKA are consistent with
previous data indicating that they have distinct and redundant
roles in filamentation .
Interestingly, cultures of cbk1D/CBK1 tpk1D/D in SM contained
significant numbers of filaments that showed characteristics of both
pseudohyphae and hyphae (Figure 5D). This hybrid morphology
was not observed in cultures of wild type, cbk1D/CBK1, or tpk1D/D
cells. Similar hyphae-pseudohyphae hybrid morphologies were
recently observed by Carlisle et al. in cells expressing an intermediate
level of UME6 , suggesting that concurrent disruption of both
RAM and PKA pathways interferes with the ability of the cell to
commit to one morphotype. These observations also suggest that a
balance between the activities of the PKA and RAM pathway is
required for normal morphogenesis.
Increased and/or dysregulated PKA pathway activity has been
linked previously to increased pseudohyphae formation. For
example, Tebarth et al. have shown that overexpression of EFG1
induces constitutive pseudohyphae . We, therefore,
hypothesized that elevated PKA activity might be responsible for the
constitutively pseudohyphal phenotype displayed by ace2D/D as
well as the increased proportion of pseudohyphae observed with
cbk1D/CBK1 heterozygotes showing CHI. Three observations
support this hypothesis. First, treatment of ace2D/D cells with the
substrate-based PKA inhibitor MyrPKI , under non-inducing
conditions, significantly increased the number of yeast-like cells
and decreased the number of mature pseudohyphae (Figure 6A),
strongly supporting the notion that increased PKA activity is
involved in the constitutive pseudohyphal phenotype of ace2D/D.
Second, EFG1 expression is elevated in both RAM pathway
mutants and cbk1D heterozygotes relative to wild type over the
Figure 6. Elevated PKA activity accounts for increase pseudohyphae in RAM mutants. (A) ace2D/D cells were incubated in YPD at 30uC for
3 h 2/+ PKA inhibitor MyrPKI (10 mM) and examined by light microscopy. (B) EFG1 expression was determined by semi-quantitative RT-PCR for each
strain at the indicated time after transfer to SM at 37uC. ACT1 levels were used as loading control. The graph indicates the fold change in EFG1 levels
for the mutant strains relative to wild type at the 180 min time point. The bars indicate the mean fold change in EFG1 relative to wild type for three
independent replicates and the error bars indicate standard deviation. The brackets indicate that the difference between EFG1 transcript levels was
statistically significant for each mutant relative to wild type (Students t test, p,0.02). (C) The expression of ENO1 and PGK1 were examined in the
indicated strains as described in Figure 5A. The brackets indicate that the difference between PGK1 transcript levels was statistically significant for the
two mutants (Students t test, p,0.02).
time course of hyphal induction (Figure 6B). Densitometric
analysis of three replicates of the 180 min time point indicates
that the EFG1 levels are 24 fold higher in each of the mutants
relative to wild type (p,0.02, Students t test). To further confirm
this elevation, we compared the levels of EFG1 in wild type and the
double heterozygote cbk1D/CBK1 pgk1D/PGK1. Consistent with
the semi-quantitative data, EFG1 is elevated in cbk1D/CBK1
pgk1D/PGK1 relative to wild type (4.8 log2, std. dev. 0.9, p = 0.01,
Students t test). Third, deletion of both alleles of EFG1 in the
cbk1D/CBK1 background decreases expression of ENO1 by a
modest 1.5-fold and PGK1 a more significant 8-fold relative to the
parental strain (Figure 6C), indicating that at least a portion of the
increased expression of putatively co-regulated genes in RAM
mutants is mediated by the PKA-Efg1 pathway. Taken together,
these experiments suggest that some of the CBK1-interacting genes
isolated in our screen are part of the transcriptional output of both
the PKA and RAM pathways and that decreased RAM function
in the CBK1 double heterozygotes leads to a compensatory
increase in PKA pathway activity which, in turn, manifests as a
phenotype of increased pseudohyphal growth due to increased
EFG1 levels .
A balance between RAM and PKA pathwzay activity is
required for normal morphogenesis
Although our results strongly suggest that the RAM and PKA
pathways interact during morphogenesis and that the PKA
pathway may be hyper-activated in the absence of RAM activity,
it remained to be determined how these pathways interact during
normal morphogenesis. As discussed above, one of the best
characterized functions of Ace2 in both S. cerevisiae and C. albicans is
as a daughter cell-specific transcription factor [26,38,39]. Two
other laboratories [26,39] have previously shown that in C.
albicans, Ace2 localizes to daughter nuclei in actively dividing
yeast-phase cells as well as in serum-induced filaments; our results
confirm those findings in SM (Figure 7A). We, therefore,
hypothesized that the relative contributions of Ace2 and Efg1 to gene
regulation during the course of hyphal development may
correspond to the timing of their nuclear localization. To our
knowledge, the nuclear localization of Efg1 during filamentation
had not been described previously.
To test this hypothesis, we used indirect immunofluorescence
to compare the proportion of cells with nuclear Efg1 at the
initiation of hyphal development to the proportion in hyphal cell
nuclei. As shown in Figure 7B, Efg1 is present in the nuclei of 50
60% (n = 100 cells) of cells prior to shifting to SM. In contrast,
Efg1 is detectable in only ,10% of hyphal nuclei.
Correspondingly, Efg1 occupancy of the promoter regions of ENO1 and
PGK1 is also higher at the initiation of hyphal development by
ChIP analysis (Figure 7C). This suggests that Efg1 may be more
important at the onset of, or early in, the filamentous transition,
while Ace2 contributes to Efg1/Ace2 co-regulated gene
transcription as daughter cell nuclei accumulate within the hyphal
Consistent with this model, ACE2 expression increases over
the 3-hour time course of hyphal induction (Figure 7D); this
finding is also consistent with its role in gene expression within
daughter cell nuclei. Interestingly, the promoter region of ACE2
has five Efg1 consensus binding sites, suggesting that the PKA
pathway may contribute to the regulation of ACE2 expression.
However, treatment with the PKA inhibitor MyrPKI reduced
levels of ACE2 expression only modestly after 3 hours in SM
(Figure 7E). Although this observation supports a possible direct
link between the PKA and RAM pathways, it suggests that
PKAEfg1 is not the sole, or even dominant, regulator of ACE2
As a whole, these data support a model in which Efg1 plays a
more important role at the initiation of hyphal development in
SM, and Ace2 plays a more important role once daughter nuclei
accumulate within the hyphal structure. Since EFG1 expression is
maintained throughout the time course of hyphal development
(Figure 6B) and Efg1 is present in some hyphal nuclei (Figure 7A),
it is unlikely that the relationship between Ace2 and Efg1
represents an either/or type of scenario. Instead, it seems more
likely that a balance exists between the relative contributions of the
two transcription factors to gene expression and that this balance
varies during hyphal development.
Methods for the large-scale genetic analysis of Candida albicans
have advanced tremendously in recent years, leading to a number
of important and informative studies . To our knowledge,
however, no large-scale synthetic genetic analyses have yet been
reported. Here, we present the first such screen. Our approach
was based on a CHI strategy, and, like other large-scale genetic
analyses of C. albicans, we employed transponson-mediated
insertional mutagenesis to generate a large collection of double
heterozygous mutants derived from a parental strain containing a
heterozygous null mutation of the RAM pathway kinase CBK1.
This library was then used to screen for genes that interacted with
CBK1 during SM-induced morphogenesis.
First and foremost, our data establishes that CHI-based genetic
interaction screening is a useful method for the genetic analysis of
the obligate diploid yeast C. albicans. A priori, CHI-based genetic
screening of a signaling network such as the RAM pathway would
be expected to identify genes that interact with the query gene
through a variety of mechanisms. Inspection of our dataset
confirms these expectations in that it includes transcriptional
targets of the RAM pathway (e.g., ENO1, PGK1), genes that likely
affect the function of pathway components (e.g., NSP1, SLK19),
and genes that function in parallel pathways (e.g., MAF1, SLF1). In
the specific case of screening a protein kinase mutant, it should
also be possible to identify substrates of that kinase. Although no
bona fide substrate of Cbk1 has been confirmed in C. albicans, our
screen identified a very likely candidate in Ssd1. Ssd1 is a well
characterized Cbk1 substrate in S. cerevisiae  and has been
shown previously to interact genetically with CBK1 in both S.
cerevisiae  and C. albicans . A consensus Cbk1
phosphorylation sequence has recently been identified in S. cerevisiae .
Supporting the possibility that CaSsd1 is a substrate of CaCbk1 is
the presence of this consensus phosphorylation sequence. Of the
remaining CBK1-interactors, RGD3, an uncharacterized potential
Rho GTPase, and VPS13, a protein involved in vacuolar protein
sorting, also have sequences that match the consensus
phosphorylation sequence for ScCbk1 (data not shown). Studies directed
towards confirming these putative Cbk1 substrates are in progress.
The CBK1-derived double heterozygous mutants isolated in our
screen displayed phenotypes indicative of defects in the
Ace2dependent functions of the RAM pathway in that they were only
observed on SM ; mutations in genes affecting
Ace2independent functions would be expected to display filamentation
defects on both SM and serum . Since many of the interacting
genes appear to be transcriptional targets of Ace2, we propose that
the effect of partially disabling the RAM pathway by deletion of
one allele of CBK1 is exacerbated by further deletion of one allele
of a gene regulated by the CBK1-dependent transcription factor
Ace2. The cumulative effect of these two mutations results in
phenotypes (increased proportion of pseudohyphae) consistent
with a further decrease in Ace2-mediated RAM transcriptional
activity. By this analysis, Ace2-transcriptional targets that display
CHI interactions with CBK1 would, therefore, appear to be
particularly important components of the transcriptional output of
the RAM pathway during morphogenesis on SM.
A particularly powerful feature of synthetic genetic analysis is the
ability to identify interactions between regulatory pathways and, in
this regard, our CHI screen of cbk1D/CBK1 was quite informative,
highlighting the interplay between the RAM and PKA pathways
during morphogenesis. Although no components of the PKA
signaling pathway were identified as CBK1-interactors, analysis of
the dataset revealed that many of the interactors were regulated by
the PKA pathway. Indeed, the similar transcriptional characteristics
of the PKA-regulated transcription factor Efg1 and Ace2 in C.
albicans have been previously noted  and, while our work was in
progress, Wang et al. reported that Efg1 and Ace2 bound to the
promoters of C. albicans genes involved in cell separation . In
addition, the PKA and RAM pathways have been linked genetically
in S. cerevisiae through experiments showing that ectopic
overexpression of the PKA kinase subunit TPK1 suppresses growth and
budding defects of RAM pathway mutants in an Ace2-independent
manner . Our data suggest that the PKA and RAM pathway
interact in C. albicans with respect to Ace2-dependent functions.
Consistent with this model, consensus binding sites for both
Efg1 and Ace2 are located in the promoter regions of a significant
proportion of CBK1-interactors. A genome-wide search identified
384 putative Efg1/Ace2 co-regulated genes, suggesting that the
two pathways interact to modulate the expression of a substantial
subset of genes. The interaction of these two pathways is further
supported by our isolation of two PKA-regulated transcriptional
modulators (MAF1 & SLF1) as CBK1 interactors as well as by the
synthetic genetic interactions between CBK1 and TPK1 observed
in our follow-up studies.
The simplest manifestation of a model in which the PKA and
RAM pathways co-regulate a set of genes would be that deletion of
either ACE2 or EFG1 results in the decreased expression of
coregulated genes. As shown in Figure 5A, this is not the case as the
expression of putatively co-regulated genes is increased in both
ace2D/D and efg1D/D mutants. This suggested that the two
pathways may compensate for one another when the other
pathway is disabled. Supporting this notion, the activity of the
PKA pathway is increased in RAM pathway mutants (Figure 5B),
and EFG1 mediates a substantial portion of the increased
expression of co-regulated genes in the absence of full RAM
pathway activity (Figure 6B). Accordingly, the level of EFG1
expression is also increased (Figure 6B) and, since inappropriately
high levels of EFG1 promote pseudohyphal growth (48), this
observation provides an explanation for the increased amounts of
pseudohyphae displayed by RAM pathway mutants.
We, therefore, propose that the increased PKA activity in RAM
pathway mutants represents a compensatory response that
maintains expression of Ace2/Efg1 co-regulated genes in the
absence of a fully functional RAM pathway. However,
constitutively elevated levels of PKA activity represent a dysregulated state
and, consequently, the expression levels of the genes are not
returned to normal but are elevated. Thus, it appears that a
balance between the activity of the PKA and RAM pathways is
required to maintain properly regulated expression of co-regulated
genes. Maintaining a balance between the activities of the two
pathways appears to be required for normal hyphal development
because: 1) loss of EFG1 leads to a failure to form filaments; 2) loss
of ACE2 leads to the accumulation of pseudohyphae; and 3)
concurrent partial disruption of both pathways leads to the
formation of filaments with characteristics of both hyphae and
pseudohyphae (Figure 5D).
If, as our results suggest, a balance between PKA and RAM
pathway-mediated transcription is required for the cell to normally
undergo filamentation, then how is this balance established and
maintained? Although further work will be required to determine
the molecular mechanism of this interaction, the cell
cycleregulated nature of both Efg1 and Ace2 suggests that the pathways
might be active at different times during morphogenesis. Ace2, for
example, localizes to the nuclei of daughter cells in both yeast and
filamentous C. albicans [26,39]. Efg1, on the other hand, has been
shown to be rapidly down-regulated soon after hyphal induction in
some conditions . These considerations led us to propose that
Efg1 may be more important in the expression of co-regulated
genes earlier in morphogenesis, while Ace2 is the dominant
regulator later in morphogenesis when daughter nuclei appear
within the filament.
Consistent with that model, we showed that more nuclei contain
Efg1 at the initiation of morphogenesis than later in the process.
Ace2, on the other hand, is absent from the vast majority of nuclei
at the initiation of morphogenesis but is found in daughter nuclei
as they accumulate within the filament (Figure 7A). Consistent
with its role later in morphogenesis, overall expression of ACE2
also increases as the cells are exposed to inducing condition for
longer periods of time (Figure 7D). Since Efg1 remains detectable
in hyphal nuclei (Figure 7B), it is unlikely that Ace2 replaces Efg1
entirely but rather Ace2 may become relatively more important as
daughter nuclei accumulate within the filament and undergo
mitosis. Thus, it seems that a balance between the PKA and RAM
pathways exists and that this balance is important for smooth
morphogenesis. A potential illustration of the importance of this
balance is provided by the morphologies displayed by the tpk1D/D
cbk1D/CBK1 mutant in which single filaments show characteristics
of both hyphae and pseudohyphae.
This model is also consistent with the observations of Wang et al.,
who reported that Efg1 represses the expression of Ace2-regulated
cell separation genes during hyphal development . They found
that in wild type strains, the Ace2-regulated expression of chitinase
CHT3 occurs approximately 3 hours post-hyphal induction, a point
at which multiple septa and daughter nuclei have formed within the
hyphal filament. The 3-hour time point also corresponds to the time
when we observed high levels of ACE2 expression. In EFG1
mutants, on the other hand, Wang et al. found that CHT3 is
inappropriately expressed within the first hour of induction and is
expressed at higher levels at 3 hours . Our observations
regarding the timing of Efg1 nuclear localization correlate well
with these expression data in that Efg1 is present early when it
suppresses Ace2-mediated CHT3 expression but is absent when
CHT3 expression is induced. It is important to note that Efg1 has
previously been proposed to function as both a transcriptional
activator and repressor during hyphal morphogenesis [39,41] and,
taken together with the observations of Wang et al., our data are
consistent with such a role.
At this point, further work will be required to understand the
molecular mechanisms by which the RAM and PKA pathway
interact. As noted above, ACE2 does possess a number of Efg1
consensus binding sites within its promoter. This suggests a
possible feed-forward mechanism by which Efg1 activates the
expression of ACE2, which, in turn, takes over transcription of
coregulated genes. However, chemical inhibition of the PKA
pathway only modestly reduced expression of ACE2 during hyphal
induction (Figure 7E). Similarly, efg1D/D mutants also exhibit very
slight changes in ACE2 expression (data not shown). Although
there may be an operative component of this feed-forward
mechanism, it seems to be a relatively minor contributor to the
crosstalk between these pathways.
In summary, we have shown that CHI-based genetic interaction
screening is a useful approach for the analysis of complex
phenotypes in C. albicans. The application of this approach to
the RAM pathway has provided insights into the mechanisms by
which the PKA and RAM signaling pathways function together
during the transition from yeast to filamentous cells in C. albicans.
Materials and Methods
Strains, media, and growth conditions
All strains are derived from CAI4 (ura3D::imm434/ura3D::imm434).
 was used as the parental strain for transposon mutagenesis. A
complete list of strains and genotypes is provided in Table S1. Yeast
peptone dextrose supplemented with 80 mg/L uridine, synthetic
dextrose medium lacking uracil, and SM were prepared using
standard recipes [15,52]. Induction of filamentation was carried out
using SM plates (37uC, 3D) or liquid SM (37uC, 3 h). All phenotypes
were confirmed on SM plates supplemented with uracil to control for
possible positional effects of URA3 expression. Proportions of yeast,
pseudohyphae and hyphae in liquid cultures were determined by
light microscopy using morphological scoring criteria described by
Sudbery et al. .
C.albicans strain WO-1 pEMBLY23 genomic DNA library (NIH
AIDS Research & Reference Reagent Program) was mutagenized
(9 independent reactions) in vitro using the GPS3-Mutagenesis
system from New England Biolabs (Beverly, MA) and a donor
plasmid (pGPS3) containing the CaURA3-dpl200 cassette 
inserted at the Spe I restriction site. Mutagenized genomic
fragments were released by PvuII digestion and transformed into
CAMM-292 using a lithium acetate-protocol with heat shock at
44uC for 20 min . The library is available upon request from
the Kumar laboratory ().
Identification of transposon insertion sites
Transposon insertion sites were amplified by 39 RACE (rapid
amplification of cDNA ends) using primers complementary to the
ends of the transposon construct, cloned into a TA vector, and
sequenced. Insertion sites were then identified by BLASTN searches
using the Candida Genome Database (www.candidagenome.
Construction of cbk1D/CBK1-derived double
Ten double heterozygotes that showed CHI were independently
constructed from the Ura- parental strain cbk1D/CBK1
(CAMM292) using fusion PCR methods to generate URA3-based
knockout cassettes . The cassettes were used to transform
CAMM292 to Ura prototrophy, and correct integration was
confirmed by PCR. Two independent isolates were evaluated for
qRT-PCR and Chromatin Immunoprecipitation assays
Total RNA was isolated using the RiboPure Yeast Kit (Ambion,
Austin, TX) and reverse transcribed using the SuperScript III First
Strand Synthesis Kit (Invitrogen, Carlsbad, CA). Changes in
transcript levels of target genes were analyzed using the Platinum
SYBR Green Mix (Invitrogen) and normalized to ACT1 levels
using the 22DDCt method . ChIP assays were performed as
described previously  using Ura+ CAI4-dervatives containing
ACE2-TAP and EFG1-MYC alleles.
Protein kinase A assay
Protein kinase A activity was measured in total cell lysates using
the PepTag cAMP-dependent protein kinase assay kit (Promega,
Madison WI) following a protocol previously developed for C.
albicans . Lysates were prepared from wild type and ace2D/D
cells that had been exposed to SM for 3 h. Phosphorylation of the
PepTag substrate was determined by agarose gel electrophoresis;
the unphosphorylated substrate migrates in the opposite direction
as the phosphorylated substrate. Images of the gel were captured
on a gel-doc imaging system and processed using Adobe
PhotoShop software. Identical contrast and levels were used for each
Light and fluorescence microscopy was performed using a Nikon
ES80 epi-fluorescence microscope equipped with a CoolSnap CCD
camera. Images were collected using NIS-Elements Software and
processed in PhotoShop. Indirect immunofluorescence was
performed as previously described using anti-Myc (Invitrogen)
primary- and TexasRed-conjugated (Molecular Probes)
secondaryantibodies . DAPI and Calcofluor white staining was performed
as described .
Table S2 Table of GO terms, number of genes per GO
category, p-values and example ORFs containing both Ace2
(MMCCASC) and Efg1 (CANNTG) binding sites within 1000 bp
of the start codon. ORFs were identified by searching the CGD
database (www.candidagenome.org) and analyzed using GO
toolbox statistical software (http://genome.crg.es/GOToolBox/).
We thank Aaron Mitchell, William Fonzi, Joachim Ernst, Joe Bliss, Judy
Berman, Carlos Vasquez, Alison Butler, and Melanie Wellington for
strains, reagents, and/or helpful discussions.
Conceived and designed the experiments: NB YC-R AK DJK. Performed
the experiments: NB YC-R TX CJ SS QS CJD MJE CPA AJB DJK.
Analyzed the data: NB YC-R TX CJ SS AK DJK. Contributed reagents/
materials/analysis tools: NB YC-R TX CJ SS QS CJD MJE CPA AJB
DJK. Wrote the paper: YC-R AK DJK.
1. Vazquez JA , Sobel JD ( 2003 ) Candidiasis . In: Dismukes WE, Pappas PG , Sobel JD, eds. Clinical Mycology . Oxford: Oxford University Press. pp 143 - 188 .
2. Lo HJ , Kohler JR , DiDomenico B , Loebenber D , Cacciapuoti A , et al. ( 1997 ) Nonfilamentous C. albicans mutants are avirulent . Cell 90 : 939 - 949 .
3. Kumamoto CA , Vinces MD ( 2005 ) Contributions of hyphae and hypha-coregulated genes to Candida albicans virulence . Cell Microbiol 7 : 1546 - 1554 .
4. Saville JP , Lazzell AL , Monteagudo C , Lopez-Ribot JL ( 2003 ) Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection . Eukaryot Cell 2 : 1053 - 1060 .
5. Noble SM , French S , Kohn LA , Chen V , Johnson AD ( 2010 ) Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity . Nat Genet 42 : 590 - 598 .
6. Whiteway M , Bachewich C ( 2007 ) Morphogenesis in Candida albicans . Annu Rev Microbiol 61 : 529 - 553 .
7. Hall RA , Cottier F , Muhlschlegel FA ( 2009 ) Molecular networks in the fungal pathogen Candida albicans . Adv Appl Microbiol 67 : 191 - 212 .
8. Tong AH , Boone C ( 2006 ) Synthetic genetic array analysis in Saccharomyces cerevisiae . Methods Mol Biol 313 : 171 - 192 .
9. Jin R , Dobry CJ , McGowan PJ , Kumar A ( 2008 ) Large-scale analysis of yeast filamentous growth by systematic gene disruption and overexpression . Mol Biol Cell 19 : 284 - 296 .
10. Dixon SJ , Costanzo M , Baryshnikov A , Andrews B , Boone C ( 2009 ) Systematic mapping of genetic interaction networks . Annu Rev Genet 43 : 601 - 625 .
11. Costanzo M , Baryshnikova A , Bellay J , Kim Y , Spear ED , et al. ( 2010 ) The genetic landscape of a cell . Science 327 : 425 - 431 .
12. Braun BR , Johnson AD ( 2000 ) TUP1, CPH1, and EFG1 make independent contributions to filamentation in Candida albicans . Genetics 155 : 57 - 67 .
13. Noble SM , Johnson AD ( 2007 ) Genetics of Candida albicans, a diploid human fungal pathogen . Annu Rev Genet 41 : 193 - 211 .
14. Bruno VM , Mitchell AP ( 2004 ) Large-scale gene function analysis in Candida albicans . Trends Microbiol 12 : 157 - 161 .
15. Uhl MA , Biery M , Craig N , Johnson AD ( 2003 ) Haploinsufficiency-based large scale forward genetic analysis of filamentous growth in the diploid fungal pathogen C . albicans. EMBO J 22 : 2668 - 2678 .
16. Oh J , Fung E , Schlecht U , Davis RW , Giaver G , et al. ( 2010 ) Gene annotation and drug target discovery in Candida albicans with a tagged transposon mutant collection . PLoS Path 6 : e1001140. doi:10.1371/journal.ppat.1001140.
17. Davis DA , Bruno VM , Loza L , Filler SG , Mitchell AP ( 2002 ) Candida albicans Mds3p, a conserved regulator of pH responses and virulence identified through insertional mutagenesis . Genetics 162 : 1753 - 1581 .
18. Epp E , Walther A , Lepine G , Leon Z , Mullick A , et al. ( 2010 ) Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation but not endocytosis . Mol Microbiol 75 : 1182 - 1198 .
19. Homann OR , Dea J , Noble SM , Johnson AD ( 2009 ) A phenotypic profile of the Candida albicans regulatory network . PLoS Genet 5 : e1000783. doi:10.1371/ journal.pgen.1000783.
20. Becker JM , Kauffman SJ , Hauser M , Huang L , Lin M , et al. ( 2010 ) Pathway analysis of Candida albicans survival and virulence determinants in a murine infection model . Proc Nat Acad Sci USA 107 : 22044 - 22049 .
21. Haarer B , Viggiano S , Hibbs MA , Troyanskaya OG , Amberg DC ( 2007 ) Modeling complex genetic interactions in a simple eukaryotic genome: actin displays a rich spectrum of complex haploinsufficiencies . Genes Dev 21 : 148 - 159 .
22. Stearns T , Botstein D ( 1988 ) Unlinked noncomplementation: isolation of new conditional lethal mutantion in each of the tubulin genes of Saccharomyces cerevisiae . Genetics 119 : 249 - 260 .
23. Vinh DB , Welch MD , Corsi AK , Wertman KF , Drubin DG ( 1993 ) Genetic evidence for functional interactions between noncomplementing (Anc) gene products and actin cytoskeletal proteins in Saccharomyces cerevisiae . Genetics 135 : 275 - 286 .
24. McNemar MD , Fonzi WA ( 2002 ) Conserved serine/threonine kinase encoded by CBK1 regulates expression of several hypha-associated transcripts and genes encoding cell wall proteins in Candida albicans . J Bacteriol 184 : 2058 - 2061 .
25. Kelly MT , MacCallum DM , Clancy SD , Odds FC , Brown AJ , et al. ( 2004 ) The Candida albicans CaACE2 gene affects morphogenesis , adherence, and virulence. Mol Microbiol 53 : 969 - 983 .
26. Mulhern SM , Logue ME , Butler G ( 2006 ) Candida albicans transcription factor Ace2 regulates metabolism and is required for filamentation in hypoxic conditions . Eukaryot Cell 5 : 2001 - 2013 .
27. Song Y , Cheon SA , Lee KE , Lee S-Y , Lee B-K , et al. ( 2008 ) Role of the RAM network in cell polarity and hyphal morphogenesis in Candida albicans . Mol Biol Cell 19 : 5456 - 5477 .
28. Nelson B , Kurischko C , Horecka J , Mody M , Nair L , et al. ( 2003 ) RAM: a conserved signaling network that regulates Ace2p transcriptional activity and polarized morphogenesis . Mol Biol Cell 14 : 3782 - 3803 .
29. Biery MC , Stewart FJ , Stellwagen AE , Ralidgh EA , Craig NL ( 2000 ) A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis . Nucleic Acids Res 28 : 1067 - 1077 .
30. Kumar A , Seringhaus M , Biery MC , Sarnovsky RJ , Umansky L , et al. ( 2004 ) Large-scale mutagenesis of yeast genome using a Tn7-derived multipurpose transposon . Genome Res 14 : 1975 - 1986 .
31. Seringhaus M , Kumar A , Hartigan J , Snyder M , Gerstein M ( 2006 ) Genomic analysis of insertion behavior and target specificity of mini-Tn7 and Tn3 transposons in Saccharomyces cerevisiae . Nuclei Acid Res 34 : e57 .
32. Wilson RB , Davis D , Enloe BM , Mitchell AP ( 2000 ) A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions . Yeast 16 : 65 - 70 .
33. Noble SM , Johnson AD ( 2005 ) Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans . Eukaryot Cell 4 : 298 - 309 .
34. Jansen JM , Wanless AG , Seidel CW , Weiss EL ( 2009 ) Cbk1 regulation of the RNA-binding protein Ssd1 integrates cell fate with translational control . Curr Biol 19 : 2114 - 2120 .
35. Jorgensen P , Nelson B , Robinson MD , Chen Y , Andrews B , et al. ( 2002 ) Highresolution genetic mapping with ordered arrays of Saccharomyces cerevisiae deletion mutants . Genetics 162 : 1091 - 1099 .
36. Ross KE , Cohen-Fix O ( 2003 ) The role of Cdh1 in maintaining genomic stability in budding yeast . Genetics 165 : 489 - 503 .
37. Havens KA , Gardner MK , Kamieniecki RJ , Dresser ME , Dawson DS ( 2010 ) Slk19 regulates spindle dynamics through two independent mechanisms . Genetics 186 : 1247 - 1260 .
38. Parnell EJ , Stillman DJ ( 2008 ) Betting a transcription factor to only one nucleus following mitosis . PLoS Biol 6 : e229. doi:10.1371/journal.pbio.0060229.
39. Wang A , Raniga PP , Lane S , Lu Y , Liu H ( 2009 ) Hyphal chain formation in Candida albicans: Cdc28-Hgc1 phosphorylation of Efg1 represses cell separation genes . Mol Cell Biol 29 : 4406 - 4416 .
40. Dihlmann MA , Herth W , Hurt EC ( 1992 ) NSP1 depletion in yeast affects nuclear pore formation and nuclear import . Eur J Cell Biol 59 : 280 - 295 .
41. Doedt T , Krishnamurthy S , Bockmuhl DP , Tebarth B , Stempel C , et al. ( 2004 ) APES proteins regulate morphogenesis and metabolism in Candida albicans . Mol Biol Cell 15 : 3167 - 3180 .
42. Willis IM , Moir RD ( 2006 ) Integration of nutritional and stress signaling pathways by Maf1 . Trends Biochem Sci 32 : 51 - 53 .
43. Robertson LS , Fink GR ( 1998 ) Three yeast A kinases have specific signaling functions in pseudohyphal growth . Proc Nat Acad Sci USA 95 : 13783 - 13787 .
44. Wolyniak MJ , Sundstrom P ( 2007 ) Role of actin cytoskeletal dynamics in activation of the cyclic AMP pathway and HWP1 gene expression in Candida albicans . Eukaryot Cell 6 : 1824 - 1840 .
45. Zou H , Fong H-M , Zhu Y , Wang Y ( 2010 ) Candida albicans Cyr1, Cap1 and Gactin form a sensor/effector apparatus for activating cAMP synthesis in hyphal growth . Mol Microbiol 75 : 579 - 591 .
46. Bockmuhl DP , Krishnamurthy S , Gerads M , Sonneborn A , Ernst JF ( 2001 ) Distinct and redundant roles of the two protein kinase A isoforms Tpk1p and Tpk2p in moprhogenesis and growth of C . albicans. Mol Microbiol 42 : 1243 - 1257 .
47. Carlisle PL , Banerjee M , Lazzell A , Monteagudo C , Lopez-Ribot JL , et al. ( 2009 ) Expression levels of a filament-specific transcriptional regulator are sufficient to determine Candida albicans morphology and virulence . Proc Natl Acad Sci USA 106 : 599 - 604 .
48. Tebarth B , Doedt T , Krishnamurthy S , Weide M , Monterola F , et al. ( 2003 ) Adaptation of the morphogenetic pathway in Candida albicans by negative autoregulation and PKA-dependent repression of the EGF1 gene . J Mol Biol 329 : 949 - 962 .
49. Cloutier M , Castilla R , Bolduc N , Zelada A , Martineau P , et al. ( 2003 ) The two isoforms of cAMP-dependent protein kinase catalytic subunit are involved in the control of dimorphism in the human fungal pathogen Candida albicans . Fungal Genet Biol 38 : 133 - 141 .
50. Ernst JF , Tielker D ( 2009 ) Response to hypoxia in fungal pathogens . Cell Microbiol 11 : 183 - 190 .
51. Schneper L , Krauss A , Miyamoto, Fang S , Broach JR ( 2004 ) The ras/protein kinase A pathway acts in parallel with the Mob2/Cbk1 pathway to effect cell cycle progression and proper bud site selection . Eukaryot Cell 3 : 108 - 120 .
52. Burke DJ , Dawson D , Stearns T ( 2000 ) Methods in Yeast Genetics . Woodville NY: Cold Spring Harbor Laboratory Press.
53. Sudbery P , Gow N , Berman J ( 2004 ) The distinct morphogenic states of Candida albicans . Trends Microbiol 12 : 317 - 324 .
54. Walther A , Wendland J ( 2003 ) An improved transformation protocol for the human fungal pathogen Candida albicans . Curr Genet 42 : 339 - 343 .
55. Schmittgen TD , Livak KJ ( 2008 ) Analyzing real-time PCR data by the comparative CT method . Nat Protoc 3 : 1101 - 1108 .
56. Xu T , Shively CA , Jin R , Eckwahl MJ , Dobry CJ , et al. ( 2010 ) A profile of differentially abundant proteins at the yeast cell periphery during pseudohyphal growth . J Biol Chem 285 : 15476 - 15488 .
57. Hnisz D , Majer O , Frohner IE , Komnenovic V , Kuchler K ( 2010 ) The Set3/ Hos2 histone deacetylase complex attenuates cAMP/PKA signaling to regulate morphogenesis and virulence of Candida albicans . PLoS Pathog 6 : e1000889. doi:10.1371/journal.ppat.1000889.
58. Kumar A , Agarwal S , Heyman JA , Matson S , Heidtman M , et al. ( 2002 ) Subcellular localization of the yeast proteome . Genes Dev 16 : 707 - 719 .