Pooled Segregant Sequencing Reveals Genetic Determinants of Yeast Pseudohyphal Growth
Kumar A (2014) Pooled Segregant Sequencing Reveals Genetic Determinants of Yeast Pseudohyphal Growth. PLoS
Genet 10(8): e1004570. doi:10.1371/journal.pgen.1004570
Pooled Segregant Sequencing Reveals Genetic Determinants of Yeast Pseudohyphal Growth
Qingxuan Song 0
Cole Johnson 0
Thomas E. Wilson 0
Anuj Kumar 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 Departments of Pathology and Human Genetics, University of Michigan Medical School , Ann Arbor, Michigan , United States of America
The pseudohyphal growth response is a dramatic morphological transition and presumed foraging mechanism wherein yeast cells form invasive and surface-spread multicellular filaments. Pseudohyphal growth has been studied extensively as a model of conserved signaling pathways controlling stress responses, cell morphogenesis, and fungal virulence in pathogenic fungi. The genetic contribution to pseudohyphal growth is extensive, with at least 500 genes required for filamentation; as such, pseudohyphal growth is a complex trait, and linkage analysis is a classical means to dissect the genetic basis of a complex phenotype. Here, we implemented linkage analysis by crossing each of two filamentous strains of Saccharomyces cerevisiae (S1278b and SK1) with an S288C-derived non-filamentous strain. We then assayed meiotic progeny for filamentation and mapped allelic linkage in pooled segregants by whole-genome sequencing. This analysis identified linkage in a cohort of genes, including the negative regulator SFL1, which we find contains a premature stop codon in the invasive SK1 background. The S288C allele of the polarity gene PEA2, encoding Leu409 rather than Met, is linked with non-invasion. In S1278b, the pea2-M409L mutation results in decreased invasive filamentation and elongation, diminished activity of a Kss1p MAPK pathway reporter, decreased unipolar budding, and diminished binding of the polarisome protein Spa2p. Variation between SK1 and S288C in the mitochondrial inner membrane protein Mdm32p at residues 182 and 262 impacts invasive growth and mitochondrial network structure. Collectively, this work identifies new determinants of pseudohyphal growth, while highlighting the coevolution of protein complexes and organelle structures within a given genome in specifying complex phenotypes.
Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its
Supporting Information files.
Funding: This work was supported by grants 1R01-A1098450-01A1 from the National Institutes of Health and 1-FY11-403 from the March of Dimes (to AK), and
grant 2 R01 CA102563 from the National Institutes of Health/National Cancer Institute (to TEW). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The budding yeast Saccharomyces cerevisiae undergoes a
pronounced growth transition in response to nitrogen limitation or
glucose limitation, forming multicellular pseudohyphal filaments that
can spread outward from a colony and/or invade the surface of a
solid growth substrate [1,2]. Yeast pseudohyphal filament formation
is a presumed foraging mechanism, accomplished through
underlying changes in cell adhesion, cell cycle progression, and budding
[1,3,4]. During pseudohyphal growth, yeast cells remain physically
connected after cytokinesis via mechanisms encompassing the
regulated expression and shedding of the flocculin Flo11p .
Cells undergoing pseudohyphal growth exhibit increased apical
growth through reorganization of the actin cytoskeleton, regulation
of polarity proteins, and delayed G2/M progression .
The molecular basis of yeast pseudohyphal growth has been studied
extensively as a model of conserved signaling pathways controlling cell
morphogenesis and polarity. Furthermore, related processes of
filamentous development in the principal opportunistic human fungal
pathogen Candida albicans are required for virulence, and signaling
pathways between the related yeasts are conserved . Classic
studies of pseudohyphal growth in S. cerevisiae have resulted most
prominently in the identification of core pseudohyphal growth
signaling modules encompassing the Kss1p mitogen-activated protein
kinase (MAPK) cascade, the cAMP-dependent protein kinase A
(PKA) pathway, and the AMP-activated protein kinase ortholog Snf1p
. The pseudohyphal growth MAPK cascade encompasses
Ste11p, Ste7p, and the MAPK Kss1p [10,14]. Kss1p phosphorylates
the Ste12p transcription factor, resulting in dissociation of the negative
regulatory Dig1p and Dig2p interactors and binding of a
Ste12pTec1p heterodimer to target promoters, such as the FLO11 promoter
. Tpk2p, a catalytic subunit of PKA, phosphorylates the Flo8p
transcription factor, promoting Flo8p binding and transcriptional
activation at the FLO11 promoter and other regulatory sites [17,24
26]. In response to glucose limitation, FLO11 transcription is
regulated by Snf1p; the Snf1p-Gal83p isoform promotes cell adhesion
during invasive filamentation by antagonizing Nrg1p- and
Nrg2pmediated repression of FLO11 [19,27].
While the central components of these signaling pathways have
been identified, the scope of the yeast pseudohyphal stress
response is broad , and the mechanisms enabling these
genes and gene products to drive pseudohyphal filamentation are
Cellular processes in eukaryotes are brought about through
the contributions of large gene sets, and a continuing
obstacle in studying these processes lies in the identification
of critical constituent genes. The yeast pseudohyphal growth
transition is an important example of a complex cellular
growth transition. During pseudohyphal growth, yeast cells
form connected chains or filaments, constituting a means of
foraging for nutrients under conditions of nitrogen and/or
glucose limitation. Yeast pseudohyphal growth has been
studied for over two decades as a model of signaling systems
controlling stress responses, cell shape, and fungal virulence.
Hundreds of genes are required for pseudohyphal growth,
however, and the critical genes that determine the
filamentous phenotype have not been elucidated. Towards this goal,
we implemented a genetic approach to identify alleles linked
with the pseudohyphal growth phenotype. These studies
identified previously unstudied variation in proteins
functioning in a complex that controls cell polarity and in a
protein of the mitochondrial inner membrane. This work
indicates that proteins in complexes and organelles have
coevolved within a given genome to yield distinct outputs
and phenotype, while highlighting the application of an
approach that is useful for the analysis of complex
phenotypes in many eukaryotes.
incompletely defined, as are the genetic determinants within this
gene set that underlie filamentation. To further dissect
pseudohyphal growth pathways, we implemented a linkage study, coupling
whole genome sequencing with pooled segregant analysis. The
results present previously unidentified genetic determinants of
yeast invasive growth and indicate the coevolution of proteins
within complexes in driving phenotype.
Pooled Segregant Analysis of Yeast Invasive
Filamentation with Deep Sequencing
For linkage analysis, we selected as parents the non-filamentous
S288C-derived strain BY4741 and the filamentation-competent
strains S1278b and SK1 [34,35]. Filamentous-form growth in
haploid strains is classically assessed using the plate-washing assay of
Gimeno et al.  to identify pseudohyphal cells that have invaded
the agar substrate. The invasive phenotype of each parent strain in
this assay is indicated in Fig. 1A. The experimental design of the
linkage study is presented in Fig. 1B. The non-invasive
S288Cderived strain was mated with each of the filamentous S1278b and
SK1 strains, and the resulting diploid strain from each cross was
sporulated. Meiotic progeny from dissected tetrads were assayed for
agar invasion by plate-washing, and spores indicating strongly
noninvasive or invasive phenotypes were pooled for subsequent linkage
analysis. Only spores resulting from complete meiosis were included
in these phenotypic pools, and intermediate filamentation
phenotypes were excluded from subsequent analysis to provide the
greatest likelihood of identifying allelic variation with a strong effect
on filament formation. Genomic DNA was extracted from each
segregant pool and subjected to high-throughput sequencing that
yielded greater than 100-fold coverage per pool.
Identification of Allelic Variants Linked with Invasive
Phenotype between a Non-filamentous S288C Derivative
From the BY4741-by-S1278b cross, 31 complete tetrads (124
spores) were screened for agar invasion, identifying 37 strongly
invasive spores and 63 non-invasive spores (Fig. 2A). The
segregant pools were sequenced, and candidate determinants of
the invasive phenotype were identified using a linkage LOD score
of greater than 3 as an arbitrarily defined cut-off. Table S1
provides a listing of these alleles, encompassing only variants that
are in protein-coding sequence and that are non-synonymous with
respect to the encoded amino acid sequence. This allele set affects
50 genes in eleven linkage blocks physically located on seven yeast
chromosomes. Figure S1 summarizes the available functional
information for this gene set.
Representative plots of non-synonymous allelic variation with
respective LOD scores are graphed in Fig. 2B for chromosomes V
and IX, highlighting the pseudohyphal growth transcription factor
gene FLO8 and the flocculin effector gene FLO11. FLO8 is a
pseudogene in S288C-derived strains , and in this analysis, the
BY4741 allelic variant containing a premature translational stop at
codon 142 of the FLO8 sequence yielded a LOD score greater
than 17 (Fig. 2B and C). The FLO11 locus exhibits fifteen allelic
changes linked with invasive growth phenotypes (Fig. 2B and C).
Previous studies identified allelic variation in FLO11 sequence
encoding amino- and carboxy-terminal regions linked with the
ability to form biofilms on the surface of wine . We recovered
these as well as additional sites of DNA sequence variation in
FLO11, with the S1278b-encoded alleles indicating linkage with
strong invasive growth. The FLO11 sequence contains an internal
repeat region that is a source of allelic variation between some
strains and colonies [38,39]; however, we did not observe a change
in the number of these repeats between BY4741 and S1278b.
Collectively, the identification of these known pseudohyphal
growth genes demonstrates the relevance of results obtained from
our pooled segregant analysis.
PEA2 Allelic Variation Impacts Cell Morphology and
MAPK Pathway Activity
To further identify important determinants of invasion, we
screened candidates from Table S1 as follows: 1) we generated
gene deletions and assayed for invasive growth phenotypes (Table
S2), and 2) for genes yielding deletion phenotypes, we generated
mutants with swapped alleles to identify genetic variants required
for invasive growth in S1278b. In particular, we focused on alleles
of genes that contributed to cell polarity, cell cycle progression, cell
morphology, and cell responses to nitrogen/carbon limitation, as
these are hallmark characteristics of filamentation.
By this approach, we identified variation in PEA2 as an
important part of the genetics underpinning invasive growth.
Pea2p localizes to sites of polarized growth as a component of a
protein complex, termed the polarisome [40,41]. PEA2 is required
for wild-type invasive growth, mating projection formation, and
bipolar bud site selection in diploids [42,43]. In the filamentous
S1278b strain, PEA2 codon 409 specifies methionine rather than
the leucine residue encoded in the S288C-derived reference
genome. The pea2-M409 allele was linked with invasive growth,
and generation of an integrated site-specific mutation
(pea2M409L) reconstituting the S288C-encoded PEA2 allele in S1278b
resulted in decreased invasive growth (Fig. 3A). Relative to wild
type S1278b, the cell morphology of the pea2-M409L mutant is
altered, exhibiting decreased elongation (Fig. 3B); over a
population of 200 cells, the percentage of pea2-M409L cells with a
length:width ratio of less than 1.5 was nearly four-fold the
corresponding percentage in a wild type strain. In addition, the
pea2-M409L mutant is impaired in Kss1p MAPK signaling
activity. The Kss1p kinase activates the Ste12p/Tec1p
transcription factor complex, which recognizes a regulatory element (FRE)
in the FLO11 promoter. The plasmid-based Pflo11-9/10-lacZ
construct contains the Ste12p/Tec1p-responsive region of the
FLO11 promoter fused to lacZ , and, by this reporter, the
pea2M409L mutant yields significantly decreased
Ste12p/Tec1pdependent transcriptional activation of FLO11 relative to
wildtype S1278b (Fig. 3C). In contrast, the pea2-M409L mutation
results in wild-type levels of a similarly designed FLO11 promoter
fusion responsive to the PKA pathway effector Flo8p (Fig. 3C) .
Genetic Variation in PEA2 Modulates Bud Site Selection
and Spa2p Binding
Under conditions of vegetative growth haploid yeast cells bud in
an axial pattern, with new buds emerging adjacent to the preceding
bud site . Haploid cells undergoing pseudohyphal growth,
however, adopt a predominantly unipolar budding pattern wherein
the first bud forms distal to the original cell division site, and
subsequent buds cluster in the distal pole [1,10]. Here, we find that
in the S1278b background the pea2-M409L mutant, corresponding
to the S288C-encoded PEA2 allele, exhibited a decrease in unipolar
budding and an increase in axial budding relative to wild type (p,
0.001), with levels intermediate between an otherwise isogenic
wildtype strain and a pea2D mutant (Fig. 3D). For this analysis, we
examined a population of invasive cells exhibiting three or more
bud scars, such that patterns of axial, unipolar, bipolar, and random
budding could be reliably distinguished [44,45]. This budding
phenotype was evident in invasive cells, but not in an equally sized
population of cells scraped from the surface of an agar plate.
Previous studies have indicated that the majority of bud sites are
distal in a pea2D mutant ; results here also indicate that the
majority of bud sites are distal in pea2 mutants, but budding pattern
analysis does indicate that Pea2p residue 409 impacts unipolar
budding in invasive haploid cells.
In the polarisome complex, Pea2p binds the scaffolding protein
Spa2p, a large coiled-coil domain-containing protein required for
polarisome function [41,46]. Here, we assessed the possibility that
allelic variation at the PEA2 locus impacts Spa2p binding, using
Protein A (ProA)-tagged Pea2p variants to recover by
co-immunoprecipitation Spa2p tagged at its amino terminus with the
hemagglutinin (HA) epitope. In the S1278b strain, the Pea2p-M409-ProA
variant recovered more HA-Spa2p than the Pea2p-L409-ProA
variant, representing the S288C-encoded PEA2 allele (Fig. 3E).
Analysis of Genetic Variation Linked with Invasion
between BY4741 and SK1
The BY4741-by-SK1 cross was implemented as described in
Experimental Procedures, and phenotypic analysis of meiotic
progeny identified 51 and 24 strongly invasive and non-invasive
spores, respectively (Fig. 4A). Subsequent deep-sequencing of the
phenotypic pools identified allelic variation linked with invasive
growth phenotypes within eleven separated locus blocks
encompassing 88 genes exhibiting non-synonymous changes and a LOD
score of greater than 4 (Table S3). A functional breakdown of
these genes is indicated in Figure S2. In this analysis, we used a
higher LOD score relative to the S1278b-by-BY4741 cross in
order to limit the number of selected allelic variants to a
manageable size for further study, as the SK1 and BY4741
genomes are more divergent (99.5% sequence identity) than the
S1278b and BY4741 genomes (99.7% identity) . Very few
allelic variants linked with invasive growth in S1278b were also
identified in SK1, aside from a few sequences near FLO8 that are
unlikely to be causative. The set of identified alleles was primarily
distinct between the two linkage studies, and deletion phenotypes
for tested genes in SK1 are indicated in Table S4.
In haploid spores from this cross, genetic variation is most
strongly linked with the invasive growth phenotype over a region of
roughly 80,000 bp on chromosome XV, encompassing SFL1
(Fig. 4B and C). SFL1 encodes a transcriptional repressor of
pseudohyphal growth that functions by binding to the FLO11
promoter, thereby blocking transcriptional activation [24,25,48
51]. Consistent with its function in repressing FLO gene expression,
deletion of SFL1 results in exaggerated invasive growth [28,32,48].
Interestingly, the SK1 strain contains an allelic variant of SFL1 with
respect to S288C-derived strains, resulting in the conversion of
codon 477 (CAA encoding glutamine) to a TAA stop codon
(Fig. 4D). This premature stop codon truncates SFL1 prior to the
sequence encoding a domain (AA 571658) that is strongly similar
to a conserved region in Myc oncoproteins . Previous studies
have identified hyperactive filamentation in a mutant of the
CEN.PK 113-7D background upon introduction of a premature
translational stop at SFL1 codon 320 (Q320-stop) . Here, we
found that allelic variation in SFL1, encompassing a premature stop
codon (C1430T, Q477-stop) in the SK1 background, is linked to the
aggressively invasive phenotype of SK1 relative to BY4741.
Allelic Variation in MDM32 Affects Mitochondrial
Morphology and Invasive Growth
In the BY4741-by-SK1 cross, allelic variation in MDM32 was
linked to invasive growth more strongly than any other identified
locus, with a LOD score of 9. As indicated in Fig. 4BD, MDM32
is found on chromosome XV, and relative to BY4741, the SK1
allele of MDM32 encodes Ser182 and Phe262 rather than Cys
and Leu, respectively. MDM32 encodes a protein complex
subunit of the mitochondrial inner membrane required for
membrane organization, the maintenance of elongated
mitochondrial morphology, and mitochondrial DNA nucleoid stabilization
. Mitochondrial function is required for pseudohyphal growth,
as filamentation-competent strains of S. cerevisiae containing a
deleted version of the mitochondrial genome are unable to form
pseudohyphae [53,54]; however, a role for Mdm32p in enabling
invasive growth remains to be identified.
To determine the effect of MDM32 allelic variation on
pseudohyphal growth, we replaced the SK1-encoded MDM32-C546/T787
allele, specifying Mdm32p-S182/F262, with BY4741-encoded
MDM32-G546/A787, specifying Mdm32p-C182/L262, in the SK1
genetic background. This allelic swap decreased invasive growth in
SK1, and agar invasion was rescued upon reintroduction of the native
SK1-encoded MDM32 allele (Fig. 5A). The SK1 mutant containing
the BY4741-encoded allele of MDM32 exhibited a more rounded cell
morphology, with the percentage of cells displaying a cell length:width
ratio of less than 1.5 increasing from 17% in wild-type SK1 cells to
68% in the SK1 mutant (Fig. 5B). Reintroduction of SK1-encoded
MDM32 recovered levels of cell elongation similar to wild type.
To assess the impact of this allelic variation on mitochondrial
function, we grew the SK1 mutant with the BY4741 allele of
MDM32 on medium containing non-fermentable glycerol as the
sole carbon source. As shown in Fig. 5C, the allele-swapped SK1
mutant grows poorly in glycerol-containing media, indicating that
oxidative phosphorylation is impaired. The structure of the
mitochondrial network is also perturbed upon introduction of
the BY4741 allele of MDM32 in the SK1 background. Using the
mitochondrion-specific MitoTracker fluorescent dye, which
diffuses passively across the plasma membrane and concentrates in
active mitochondria by membrane potential, we can visualize a
compact and collapsed mitochondrial network in SK1 cells
containing the BY4741-encoded Mdm32p-C182/L262 variant,
similar to that observed in mdm32D (Fig. 5D). MDM32 is a
paralog of MDM31, and the encoded proteins have been found to
interact, albeit transiently and weakly, as components of protein
complexes at the mitochondrial inner membrane . We,
therefore, assessed the effect of allelic variation at MDM32 on
Mdm31p binding; however, we observed no difference in the
recovery of Mdm31p by co-immunoprecipitation between the
respective Mdm32p variants (Figure S3). In sum, MDM32 is a
determinant of invasive growth, and replacement of the native
SK1 allele of MDM32 with the BY4741-encoded allele yields a
mutant filamentous growth phenotype.
The linkage analysis presented here identifies a broad gene set
contributing to pseudohyphal growth (Fig. 6). The pattern of
allelic linkage indicates a large number of determinant loci
underlying invasive growth, consistent with results from systematic
single-gene deletion and overexpression studies [28,31,32]. Within
this gene set, components of the polarisome and mitochondria play
important roles in enabling invasive growth. We report here that
Pea2p residue 409 impacts bud site selection in haploid cells
undergoing invasive growth, although the effect is less pronounced
in determining initial distal-versus-proximal budding in virgin
mother cells. Pea2p residue 409 lies distinct from the Pea2p
coiledcoil region between residues 236 and 327 and is important for
Spa2p binding. Spa2p interacts with Ste11p and Ste7p from the
Kss1p MAPK pathway, providing a mechanism for
polarisomemediated regulation of Kss1p MAPK activity . Our results
further indicate that nuclear-encoded Mdm32p is required for
invasive growth in SK1, and that residues 182 and 262, located
outside of the mitochondrial pre-sequence (AA 1102) and at the
boundary or outside of a transmembrane domain (AA 161184
and 636653), are important in enabling invasion, as well as in
determining aerobic respiratory function and mitochondrial
morphology. Mdm32p is proposed to function cooperatively with
other inner membrane proteins and components of the outer
mitochondrial membrane in the maintenance of mitochondrial
morphology , potentially through cytoskeletal interactions that
may be affected by variation at these sites.
This analysis highlights two additional points. First, the
nonfilamentous BY4741 background is not uniformly repressive with
respect to pseudohyphal growth. From the S1278b and SK1
crosses, we identified three and five blocks of allelic variation,
respectively, in BY4741 linked with the invasive growth
phenotype; full listings of the encompassed alleles with respect to each
cross are presented in Tables S5 and S6. Alleles in S288C-derived
strains that promote pseudohyphal growth are antagonized by
alleles that repress filament formation, such as the pseudogene
form of FLO8; similarly, alleles in SK1 linked with the
noninvasive phenotype may be offset by alleles that promote invasion,
such as the SFL1 allele containing a premature stop codon.
Second, the identified allelic variation in PEA2 and MDM32 and
the allele-swapping experiments performed here indicate that
within a given genome, functionally interacting genes coevolve to
impact phenotype. The majority of genetic variation linked with
invasive phenotype in this study involves site-specific changes that
do not create pseudogenes. Alleles of these genes yield functional
proteins within the respective genomic contexts; however, a given
allele results in a hypomorphic phenotype when introduced into a
non-native strain. It should be noted that the BY4741-encoded
allele of PEA2 may be viewed as being pseudohyphal competent,
as Liu et al.  reported that the introduction of
S1278bencoded FLO8 in a S288C-derived strain is sufficient to enable at
least some degree of pseudohyphal growth. The data here suggest
that partner genes have likely co-evolved with genes such as PEA2
and MDM32, and the resulting protein complexes are, thus, an
important determinant of cell phenotype. These findings highlight
the utility in studying these complexes as a whole, in supplement to
individual proteins, in order to accurately identify the functions
and properties that specify phenotype.
It is interesting that the studies here indicated very little overlap
between alleles linked with invasive growth in the S1278b and
SK1 strains with respect to BY4741 (Fig. 6). Studies mapping
quantitative trait loci (QTL) in a cross of the laboratory strain
BY4716 and the vineyard strain RM-11 identified hotspots
impacting gene expression, protein abundance, and small
molecule-dependence . These hotspots were principally due to
alleles in the BY4716 background, leading Ronald and Akey 
to suggest that the causative polymorphisms may occur at low
frequency. The non-overlapping allele sets identified in our
analysis are not suggestive of hotspots, but rather highlights the
substantial importance of epistatic interactions in determining the
sum filamentous phenotype resulting from variant alleles in the
haploid segregants. These epistatic interactions likely represent
instances of gene coevolution, which has been suggested to occur
at an elevated rate for genes encoding proteins of shared biological
functions and/or for proteins that have coevolved between species
[60,61]. Clark et al.  have analyzed the rate of covariation for
pairs of proteins over evolutionary time, and by this analysis,
Figure 6. Chromosomal location of genes exhibiting allelic linkage with invasive growth from the BY4741 cross with S1278b and
SK1, respectively. Genes within a chromosomal region are listed in the boxes with color-coding to indicate the respective cross from which the
linkage was identified. Hash marks indicate 100,000 bases, and the full length of each chromosome is indicated.
polarisome components as a whole do not exhibit statistically
significant evidence of covariation, although many mitochondrial
complexes do yield signature indicating evolutionary rate
covariation. Further analyses of individual protein pairs from the strains
used in our study will be necessary to identify a set of coevolved
proteins that drive the filamentous growth phenotype.
In summary, we used pooled segregant whole-genome
sequencing to dissect gene networks that determine yeast pseudohyphal
growth. This analysis identified allelic variation in the known
pseudohyphal growth genes FLO8 and FLO11, while also
revealing variation in the negative regulator SFL1, the coding
sequence of which contains a premature stop codon in the invasive
SK1 background. We further found that amino acid 409 in the
polarisome protein Pea2p is a site of allelic variation critical for the
proteins ability to signal through the Kss1p MAPK pathway,
establish unipolar budding during pseudohyphal growth, and bind
the Spa2p polarisome scaffold. Linkage analysis identifies variation
in MDM32 as a determinant of invasive growth between S288C
derivatives and the SK1 strain; the 182 and 262 residues are sites
of variation and contribute to Mdm32 function in aerobic
respiration and invasive growth.
Materials and Methods
Yeast Strains and Growth Conditions
A listing of yeast strains and plasmids used in this study is
provided in Tables S7 and S8. Haploid deletion mutants were
constructed by PCR-mediated gene disruption using
pFA6aKanMX6 or pUG72 [62,63]. Yeast strains were propagated on
rich YPD medium (1% yeast extract, 2% polypeptone and 2%
glucose) medium or synthetic medium as described . Yeast
invasive growth was assayed on YPD medium.
Pooled Segregant Analysis
The statistical modeling used to derive the probabilities of
identifying linkage are described by Birkeland et al. .
Following mating as indicated in Fig. 1, resulting strains were
sporulated and asci were dissected. The dissected spores were
grown overnight at 30uC and were individually tested for mating
type. Spores resulting from complete meiosis (four viable spores
with two each of the a and a mating types) were then used for
whole genome sequencing. Each spore was assigned to invasive
or non-invasive pools based on its invasive growth phenotype.
To ensure equal representation of all segregants in a pooled
population, each haploid strain was grown overnight at 30uC in
individual 4 ml YPD cultures. The OD600 of the cultures was
determined and used to calculate the appropriate volume of each
strain so that upon mixing, we would achieve equal numbers of
cells. A mate-pair library with 300-bp fragments was prepared
for each of the phenotypic pools, and each library sequenced as
paired-end reads using the Illumina Genome Analyzer
(University of Michigan DNA Sequencing Core). Sequence analysis was
performed as described . To obtain an estimate of the
recombinant and non-recombinant spore counts in each
phenotypic pool for a given observed sequence variant, we
multiplied the number of spores in the pool by the fraction of
sequence reads from that pool that matched the corresponding
allele variant. These values were then used in standard LOD
Analysis of Yeast Budding Pattern
Budding patterns of invasive cells were determined as
previously described [44,45]. In brief, equal concentrations of
mid-log phase cultures were spotted onto YPD plates and
incubated for 7 days at 30uC; surface cells were subsequently
washed off under a gentle stream of water. Residual invaded cells
were recovered from the agar using a sterile toothpick. Cells were
washed twice in sterile water and were stained with 2 mg/ml
calcoflour white. Bud scars were visualized by fluorescence
microscopy. Cells with more than three bud scars were examined.
Budding patterns were determined by criteria previously
described . Budding patterns were divided into four
subgroups: axial, bipolar, unipolar and random. The axial pattern
was defined as a long chain of bud scars on the proximal cell pole.
Cells with a cluster of scars exclusively at the distal pole were
classified as exhibiting unipolar budding. A pattern of medial bud
scars was scored as random budding, whereas cells with bud scars
equally distributed on both proximal and distal poles were
classified as undergoing bipolar budding. For these analyses, 200
250 cells from each strain were scored.
Plasmid construction. For expression of S1278b alleles in
null mutants, yeast open reading frames of the Y826 background
with 1 kb of upstream sequence and 300 bp of downstream
sequence were cloned into a Gateway vector by standard methods
of recombination-based Gateway cloning (Invitrogen) [66,67]. For
fusing enhanced green fluorescent protein (eGFP) and tandem
affinity purification (TAP) cassettes to the carboxyl-terminus of the
budding yeast Mdm31p and Mdm32p proteins, Gateway plasmids
416-GPD-ccdB-EGFP and 414-GPD- ccdB-TAP were used .
Plasmid pCu-Spa2p-3xHA-URA3 was modified from
pCu-3xHAURA3 using standard restriction enzyme digestion and
ligationbased techniques. The SalI and HindIII restriction sites of this
plasmid was used to integrate the SPA2 open reading frame
between the copper-inducible CUP1 promoter and 3xHA/GFP
Integrated point mutations. Allelic variants of PEA2 were
generated in the S1278b background as integrated point mutations.
A URA3 cassette was amplified from the pUG6 plasmid and then
used to replace the PEA2 open reading frame in Y826.
Subsequently, 5-FOA-mediated counter selection  was applied
to replace the integrated URA3 cassette with a 500 bp cassette
encompassing the PEA2 allelic change (M409L), recreating the
S288C-derived allele in the S1278b background. The integrated
point mutation was confirmed by sequencing of an amplified PCR
fragment using the University of Michigan Sequencing Core.
Yeast invasive growth assays. Invasive growth of haploid
strains was determined by the standard plate washing assay of
Gimeno et al. . Mid-log phase cultures were spotted onto YPD
plates and incubated for 7 days at 30uC, and surface cells were
subsequently washed off under a gentle stream of water. Residual
cells from the spotted culture on the plate were imaged using a
Nikon Eclipse 80i upright fluorescence microscope.
FRE-lacZ assays. Plasmids pLG669-Z FLO11-6/7 and -9/
10 were transformed into designated yeast strains by standard
protocols . Cells were grown in SC-Ura media overnight, and
then inoculated into fresh media to an OD600 of approximately
0.2. Cultures were subsequently grown for 34 hours to an OD600
of approximately 0.81.0. b-galactosidase activity was determined
with the use of ortho-nitrophenyl-b-galactoside as a substrate
(Sigma Aldrich) [70,71].
Assays for respiratory activity. Respiratory ability was
assessed using YPG plates with glycerol as a non- fermentable
carbon source. Cells were pre-grown overnight and then inoculated
into fresh SC medium to an OD600 of approximately 0.2; culture
were subsequently incubated an additional 3 hours to an OD600 of
approximately 0.8. The same amount of cells were serially diluted
and spotted onto YPD and YPG (1% yeast extract, 2% polypeptone
and 2% glycerol) plates, and incubated for 2 days at 30uC.
Fluorescence microscopy and image processing. For
live-cell imaging, spotted cultures were grown in YPD plates for
7 days at 30uC. After washing the surface of the plates with a
gentle stream of water, cells that had invaded the agar were
recovered using a steel scalpel, and the extracted cells were washed
twice with distilled water. Images were taken using a Nikon Eclipse
80i upright fluorescence microscope. To determine budding
patterns, cells were stained with Calcofluor White as previously
described . Over 250 cells were counted twice for each strain.
Statistics were analyzed using the ANOVA approach. To reveal
mitochondrial morphology, living cells were stained with 50 nM
MitoTracker Red, and the staining pattern was visualized by
fluorescence microscopy .
Immunoprecipitation studies. PEA2 alleles were
generated by tagging with ProA on the C-terminus in wild type and
pea2M409L mutants in the S1278b strain. The SPA2 open reading
frame was cloned into pCu-3HA-URA, such that SPA2 was
expressed from a copper-inducible promoter as a fusion protein
with three copies of the hemagglutinin epitope at its amino
terminus. The resulting plasmids pCu-3HA-ScSPA2 and
pCu3HA-SSPA2 were introduced by transformation into both wild
type and pea2-M409L mutants. The binding affinity between
selected proteins was detected by co-immunoprecipitation. For
native immunoprecipitation, 2 OD units of cells were lysed in 1 ml
lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 2 mM
EDTA, 0.5% Triton X-100, 1 mM PMSF, and Complete
EDTAfree protease inhibitor [Roche]) with glass beads. After
centrifugation at 13,000 g for 10 min, the resulting supernatant was
incubated with protein G-Sepharose 4 Fast Flow (GE Life Tech)
for 2 hr at 4uC. After washing the Sepharose with lysis buffer six
times, the bound materials were eluted by boiling the Sepharose in
SDS-PAGE loading buffer. The resulting eluate was analyzed by
Western blotting with designated antibodies (anti-HA, anti-Protein
A and anti-GFP). Blots were developed using the SuperSignal
West Dura Extended Duration Substrate (Thermo Scientific).
Figure S1 Functional characterization of genes exhibiting allelic
linkage with invasive growth in the BY4741 cross with S1278b.
Genes from the analysis that have been previously identified as
exhibiting filamentous growth phenotypes upon deletion  are
listed to the left. The remaining genes are listed to the right, with
genes of unknown functions and genes with functions that may be
related to pseudohyphal growth indicated separately. Functions
are drawn from data deposited in the Saccharomyces Genome
Database as of publication. The chromosome in which the gene is
located is indicated in parentheses. It should be noted that this list
encompasses genes that may only exhibit linkage because of their
close proximity to an important allelic determinant.
Figure S2 Functional characterization of genes exhibiting allelic
linkage with invasive growth in the BY4741 cross with SK1. Genes
from the analysis that were identified as exhibiting filamentous
growth phenotypes upon deletion  are listed to the left. The
remaining genes are listed to the right, with genes of unknown
functions and genes with functions that may be related to
pseudohyphal growth indicated separately. Functions are drawn
from data deposited in the Saccharomyces Genome Database as of
publication. The chromosome in which the gene is located is
indicated in parentheses. It should be noted that this list
encompasses genes that may only exhibit linkage because of their
close proximity to an important allelic determinant.
Figure S3 Binding of Mdm31p by BY4741- and SK1-encoded
Mdm32p variants. The MDM32 open reading frames from SK1
and BY4741 were cloned into a GPD-eGFP Gateway plasmid,
such that the MDM32 sequence was expressed from the GPD1
promoter as an in-frame 39-fusion to sequence encoding enhanced
GFP. MDM31 was cloned into the GPD-TAP plasmid, yielding a
fusion of the tandem affinity purification (TAP) tag to the carboxy
terminus of Mdm31p upon expression from the GPD1 promoter.
The resulting plasmids were transformed into mdm32D mutants.
The binding affinity between Mdm32p-GFP and Mdm31p-TAP
was revealed by IgG pull-down using Mdm31p-TAP as bait. No
significant difference in binding between Mdm31p-TAP and the
respective Mdm32p-GFP variants was observed.
Table S1 Genes exhibiting allelic linkage (LOD.3) from the
BY4741-by-S1278b cross. Alleles in a single chromosome are
demarcated by a double line; groupings of alleles within a possible
linkage block are separated by a single line.
Table S3 Genes exhibiting allelic linkage (LOD.4) from the
BY4741-by-SK1 cross. Alleles grouped in a single chromosome
are indicated by a double line; groupings of alleles within a single
linkage block are indicated with a single line.
Table S6 Alleles with variation between BY4741 and SK1 where the
BY4741-encoded allele exhibits linkage with the invasive phenotype.
Alleles within a given chromosome are separated by a double line.
Strains used in this study.
Plasmids used in this study.
We thank Robert H. Lyons from the University of Michigan DNA
Sequencing Core and Shanda R. Birkeland for technical assistance. We
thank Daniel J. Klionsky for reagents and helpful discussions regarding this
Conceived and designed the experiments: QS CJ TEW AK. Performed the
experiments: QS CJ. Analyzed the data: QS CJ TEW AK. Contributed
reagents/materials/analysis tools: QS CJ. Contributed to the writing of the
manuscript: QS TEW AK.
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