Department of Genetics, University of Melbourne, Victoria 3010, Australia
* Author for correspondence (e-mail: alex.a{at}unimelb.edu.au)
Accepted 11 December 2002
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Summary |
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Key words: RAC, Penicillium marneffei, Polarization, Fungal pathogen, Actin
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Introduction |
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Of significant interest is the role of the Rho-family GTPase Rac because,
unlike the other Rho GTPases, Rac orthologs are not present in either
Saccharomyces cerevisiae or Schizosaccharomyces pombe and
very little is known about the specific role played by this GTPase. In higher
eukaryotes, a general role of Rac proteins is to regulate cellular
morphogenesis (Johnson, 1999).
Inappropriate function of the RAC homolog in Drosophila
melanogaster affects morphogenesis by resulting in defects in axon
initiation and elongation during nervous-system development, duplication of
wing hairs during wing-hair development, loss of photoreceptor differentiation
during eye development and randomized ommatidial rotation and misorientation
(Eaton et al., 1995
;
Fanto et al., 2000
;
Luo et al., 1994
).
Defects in morphogenesis in Rac mutants of some organisms have
been shown to be a direct consequence of the loss of correct actin
organization. Expression of the dominant-negative form of the Arabidopsis
thaliana Rac homolog in tobacco (Nicotiana tabacum) results in a
reduction of actin bundles and inhibition of pollen-tube elongation. In the
constitutively active form, the mutant protein leads to cellular
depolarization with excessive actin cables
(Kost et al., 1999). In
mammalian cells, the formation of actin-rich cell extensions termed
lamellipodia is dependent on induction by Rac. The dominant activated protein
induces the formation of lamellipodia, whereas the dominant-negative form
blocks lamellipodia formation (Nobes and
Hall, 1995
).
Fungi represent a highly amenable eukaryotic system in which to study
polarized growth and development. Penicillium marneffei is an
opportunistic human pathogen that is dimorphic, capable of growing in a
filamentous multinucleate hyphal form at 25°C or as a uninucleate
pathogenic yeast form at 37°C. Upon switching from hyphal growth at
25°C to yeast growth at 37°C, P. marneffei undergoes a
process termed arthroconidiation. Double septa (cross walls) are laid down
along the arthroconidiating hyphae and fragmentation occurs along the septal
plane to generate single cells. Liberated yeast cells are uninucleate and
divide by fission (Chan and Chow,
1990). Switching from yeast growth at 37°C to hyphal growth at
25°C requires the polarized growth of yeast cells, septation, branching
and the uncoupling of nuclear and cellular division to produce multinucleate
cells, which form the multinucleate hyphal mycelium characteristic of P.
marneffei at 25°C. The two growth forms possess unique modes of
polarized growth. In addition to dimorphic growth, P. marneffei also
undergoes an asexual developmental program at 25°C. This complex
developmental process requires the differentiation of multiple specialized
cell types that form a structure termed a conidiophore. This differentiation
process culminates in the production of uninucleate asexual spores (conidia)
from highly specialized sporogenous cells. Asexual development also requires
changes in polarized growth that are distinct from those required for hyphal
or yeast growth.
Previously, we identified and characterized the P. marneffei CDC42
homolog, cflA (CDC42-like gene A). Correct CflA function is
required for polarized growth of vegetative hyphae at 25°C and yeast cell
morphogenesis at 37°C. By contrast, cflA was not involved in
asexual development at 25°C and no other regulator of polarized growth
during asexual development was known (Boyce
et al., 2001). During the cloning of cflA, a second
related gene was identified, designated cflB, which has been found to
encode another Rho GTPase, with specific homology to the RAC class.
There has been no functional characterization of a RAC homolog in a
morphologically complex fungal species to date. The RAC homolog
YlRAC1 has been cloned from the dimorphic yeast Yarrowia
lipolytica, and deletion results in defects in cell morphology and
impairment of hyphal growth (Hurtado et
al., 2000
). In this study, we have investigated the role of the
RAC homolog cflB in the growth and development of P.
marneffei. CflB is localized to plasma and internal membranes, and to
septa in live cells, and colocalizes with actin at nascent septation sites and
at the hyphal apex. By generating cflB gene deletion and
cflB dominant-negative and dominant-activated strains, we have shown
that CflB is required for the morphogenesis of conidiophores during asexual
development in both P. marneffei and Aspergillus nidulans
and show that the mechanisms regulating polarized growth in these two fungal
species differ. CflB also plays an overlapping but distinct role to CflA in
polarized growth during vegetative hyphal growth. We also show that CflB is
required for correct actin localization in vegetative and asexual cells at
25°C.
Of significant interest, is the discovery that, unlike the CDC42 homolog cflA, cflB is required for cellular polarization during asexual development but is not involved in yeast cell production at 37°C. Our results indicate that, although small GTPases play similar roles in morphogenesis and actin organization, each protein has a specialized and distinct role during development.
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Materials and Methods |
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Cloning and plasmid construction
A P. marneffei genomic library was constructed by ligating
partially Sau3AI-digested P. marneffei genomic DNA into
BamHI-digested GEM-11 and screened at low stringency (50%
formamide, 2x SSC, 37°C) for Rho GTPase homologs using P.
marneffei cflA (a CDC42 homolog). A weakly hybridizing clone was
isolated and a 3.5 kb SacI/EcoRI-hybridizing genomic
fragment was cloned into pGEM3ZF to generate pKB4811. Sequencing was performed
by the Australian Genome Research Facility and sequence was analysed using
SequencherTM 3.1.1 (Gene Codes). The Genbank accession number of the
P. marneffei cflB gene is AF515698. Database searches and sequence
comparison were performed using the Australian National Genomic Information
Service. A cDNA library, constructed using the SMART cDNA Library construction
kit (Clontech Laboratories) with RNA isolated from a 37°C vegetative
culture (Borneman et al., 2000
)
was probed with a 2.5 kb SpeI/EcoRI fragment of
cflB (50% formamide, 0.1x SSC, 37°C).
The cflB deletion construct (pKB4947) was generated by replacing
the EcoRV/SalI fragment (+183 bp to +308 bp relative to the
start codon) of the SpeI/EcoRI cflB subclone
pKB4883 with a 2.3 kb SmaI/XhoI fragment containing the
A. nidulans pyrG+ selectable marker. The cflB
gene (-182 to +813) was amplified by PCR using the primers F79 (5'-GAG
GTA CCT GAG CAT TTA TAC GGG TG-3') and F80 (5'-GAG GAT CCC ATA TAC
GCA GTA GGA TAG-3'), digested with KpnI/BamHI and
ligated into the pAL3 vector containing the A. nidulans alcA promoter
(May, 1987) to yield the
alcA(p)::cflB overexpression construct (pKB5021). To generate the
alcA(p)::cflBD123A construct, the D123A mutagenic primer
J21 (5'-CAC TAA GCT TGC TTT GAG AG-3') and the M13-20 forward
primer were used to amplify from +515 to +1593 of cflB, from which a
0.64 kb HindIII fragment containing the mutation was used to replace
the equivalent fragment in a alcA(p)::cflB subclone (pKB5021),
generating pKB5266. To generate the alcA(p)::cflBG18V
construct, the G18V mutagenic primer J20 (5'-GGT GAC CGG TGA TGT TGC
TG-3') and the M13-20 forward primer were used to amplify from +98 to
+1593 of cflB, from which a 0.86 kb AgeI fragment containing
the mutation was used to replace the equivalent fragment in a
alcA(p)::cflB subclone (pKB5021) generating pKB5263. The integrity of
the constructs was confirmed by sequencing. The cflB gene, from -1 to
+813 was amplified by PCR using the primers J55 (5'-CGA GCT CAA TGG CGT
CTG GGC-3') and F80 (5'-GAG GAT CCC ATA TAC GCA GTA GGA
TAG-3'), digested with EcoCRI/BamHI and the 0.9 kb
fragment ligated into the SmaI/BamHI-digested pAA4240 vector
containing the constitutive gpd promoter and GFP to yield
the gpdA(p)::gfp::cflB construct (pKB5441).
Fungal strains and media
P. marneffei strains FRR2161, SPM3 and SPM4, and the DNA-mediated
transformation process have been described previously
(Borneman et al., 2001). The
gpdA(p)::gfp::cflB strains were generated by co-transformation of
SPM4 with pKB5441 [gpdA(p)::gfp::cflB] and the pyrG
selectable plasmid pAB4342. The alcA(p)::cflB,
alcA(p)::cflBG18V and alcA(p)::cflBD123A
strains were generated by transformation of SPM4 with pKB5021
[alcA(p)::cflB], pKB5266 [alcA(p)::cflBD123A] or
pKB5263 [alcA(p)::cflBG18V] and selecting directly for
pyrG+. The
cflB::pyrG+ strain was
generated by transforming SPM4 with linearized pKB4947 and selecting for
pyrG+. Transformants were then screened by Southern-blot analysis.
The
cflB::pyrG- strain was isolated by growth of
the
cflB::pyrG+ strain on 5-fluororotic acid
(5-FOA) medium containing uridine and uracil to select for loss of the
pyrG marker. A 5-FOA-resistant sector was isolated and had a
restriction map consistent with loss of pyrG at the cflB
locus. This strain is unable to grow in the absence of uridine (5 mM) and
uracil (5 mM).
The A. nidulans strains were generated by transformation with pKB5021 [alcA(p)::cflB], pKB5266 [alcA(p)::cflBD123A] and pKB5263 [alcA(p)::cflBG18V] and selecting directly for pyrG+.
At 25°C, strains were grown on ANM (Aspergillus nidulans
medium) supplemented with 10 mM -amino butyric acid (GABA) as a sole
nitrogen source (Cove et al., 1966). At 37°C, strains were grown on either
brainheart infusion (BHI) or synthetic dextrose (SD) medium. The
gpdA(p)::gfp::cflB strains were grown in liquid SD medium at 37°C
(Ausubel et al., 1994
). The
alcA(p)::cflB, alcA(p)::cflBD123A and
alcA(p)::cflAG18V strains were grown on carbon-free medium
(CF) (Cove et al., 1966) with GABA and fructose (uninduced conditions) or on
CF with GABA, fructose and cyclopentonone (induced conditions).
Microscopy
P. marneffei strains were grown on slides covered with a thin
layer of solid medium and with one end resting in liquid medium
(Borneman et al., 2000).
At 25°C, alcA(p)::cflB, alcA(p)::cflBD123A and
alcA(p)::cflAG18V strains were grown in liquid CF medium
with 0.1% glucose and 10 mM GABA for 1 or 2 days, and induced by replacement
of the medium with CF medium containing 0.1 M cyclopentonone and 0.1%
fructose, and incubating for an additional 1 or 2 days. The
cflB::pyrG strain was grown on ANM supplemented with GABA at
25°C for 2 or 4 days.
At 37°C alcA(p)::cflB, alcA(p)::cflBD123A and
alcA(p)::cflAG18V strains were grown for 4 days at
37°C on slides coated with BHI and resting in BHI liquid medium before
replacement of the medium with 0.1 M cyclopentonone and 0.1% fructose for an
additional 2 days. The cflB::pyrG strain was grown on BHI
medium for 4 days at 37°C.
Immunofluorescence microscopy for detection of the actin cytoskeleton was
performed using standard protocols
(Fischer and Timberlake,
1995). Mouse C4 monoclonal anti-actin antibody (Chemicon
International) diluted at 1:200 was the primary antibody and ALEXA 488 rabbit
anti-mouse antibody (Molecular Probes) at a 1:1000 dilution was the secondary
antibody. Immunofluorescence microscopy for detection of the actin
cytoskeleton and CflB was performed using standard protocols on two
gpd(p)::gfp::cflB strains
(Fischer and Timberlake,
1995
). Mouse C4 monoclonal anti-actin antibody (1:200 dilution)
and rabbit polyclonal anti-green-fluorescent-protein (GFP) antibody (1:50
dilution) were the primary antibodies and ALEXA 488 rabbit anti-mouse antibody
(1:1000 dilution) and ALEXA 594 goat anti-rabbit antibody (1:500 dilution,
Molecular Probes) were the secondary antibodies. The FRR2161 wild-type strain,
the gpd(p)::gfp::cflB strains and the
cflB strain
were grown on ANM plus GABA slides for 4 days at 25°C and the FRR2161 and
the
cflB strain also grown on SD slides for 4 days at
37°C.
Slides were examined using differential interference contrast (DIC) or epifluorescence optics for GFP, staining with fluorescent brightener 28 (calcofluor; CAL) and 4'6-diamidino-2-phenylindole staining (DAPI) and viewed on a Reichart Jung Polyvar II microscope. Images were captured using a SPOT CCD camera (Diagnostic Instruments) and processed in Adobe PhotoshopTM 5.0.
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Results |
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The predicted protein sequence of CflB contains the domains previously
shown to be necessary for GTPase function. These include the proposed
GTP-binding and hydrolysis domains (GDGAVGKT and DTAGQE), a GDP to GTP
exchange domain (TKLD), a domain for effector interaction (TVFDNY), and a C
terminal CAAX motif (where A indicates an aliphatic amino acid and X any amino
acid), which is predicted to allow association with the plasma membrane after
prenylation (Ohya et al.,
1993; Ziman et al., 1991; Chen
et al., 1993
).
Cellular localization of CflB
A gpdA(p)::gfp::cflB fusion construct (pKB5441) was generated and
co-transformed with the pAB4342 (pyrG) selectable plasmid into strain
SPM4. Southern-blot analysis of DNA from four GFP fluorescent positive
transformants showed that they had copy numbers of between 6 and 11 (data not
shown). After 2 and 4 days of growth at 25°C, the fusion protein was
clearly localized around the entire cell periphery, at internal membranes and
at septa in both vegetative hyphae (Fig.
1A,B) and the asexual development structures (data not shown) of
all cells. In yeast cells, after 4 days at 37°C, the GFP::CflB fusion
protein was also localized around the cell periphery, at internal membranes
and at septa in all cells (data not shown). To investigate the timing of CflB
localization to septation sites, the GFP::CflB fusion protein and actin were
localized in these strains using immunocytochemistry with anti-actin and
anti-GFP antibodies. During hyphal growth, after 4 days at 25°C, actin was
localized as cortical spots along the length of hyphae and concentrated at the
hyphal apex (Fig. 1C). Actin
also formed structures at nascent septation sites
(Fig. 1D). These structures
constricted as the cell wall was laid down and actin was not detectable in
mature septa. The GFP::CflB fusion protein co-localized with actin at nascent
septation sites (Fig. 1D). In
this case, the GFP::CflB fusion protein also co-localized with actin at the
hyphal apex, presumably because of the increased sensitivity of immunostaining
(Fig. 1C).
|
Expression of cflB
Northern-blot analysis revealed that cflB produced three
differently expressed transcripts of approximately 1.0 kb, 1.1 kb and 1.3 kb
in size (Fig. 2A). Both the 1.1
kb and the 1.3 kb cflB transcripts were expressed under all tested
conditions, whereas the 1.0 kb transcript was only expressed in vegetative
hyphae and yeast cells. The 1.1kb transcript was the most abundant transcript
under all conditions and the levels were higher at 37°C than 25°C.
|
RNA was also isolated from yeast cells grown for 8 days at 37°C then transferred to 25°C and incubated for 6 hours, 18 hours or 24 hours. During this time course, P. marneffei undergoes the yeast to hyphal dimorphic transition. Northern-blot analysis showed that the levels of the 1.1 kb and 1.0 kb cflB transcripts initially increased and then decreased (Fig. 2B), indicating that cflB undergoes a modest increase in expression during the morphological changes accompanying the 37°C to 25°C dimorphic switch.
Disruption of cflB function
A deletion strain was generated by transformation of P. marneffei
with the targeted deletion construct pKB4947, in which sequences encoding
amino acids 28-52 of CflB were replaced with the A. nidulans pyrG
selectable marker. PyrG+ transformants were screened by
Southern-blot analysis and two strains were identified that showed genomic
alterations consistent with replacement of the cflB locus with the
pyrG deletion construct.
At 25°C, wild-type P. marneffei colonies consist of vegetative
hyphae, aerial hyphae and conidiophores. The colonies are green because of the
presence of asexual spores (conidia) produced by conidiophores. The
cflB strain produced abundant aerial hyphae, resulting in a
fluffy phenotype and, although these colonies clearly produced conidia (the
colony surface appeared green), they lacked visible conidiophore structures
(Fig. 3A). To confirm that the
phenotypes of the
cflB strain were due to the disruption of
cflB and not to an unlinked mutation, a
cflB::pyrG- strain was isolated by counterselection
against pyrG+ on 5-fluoroorotic acid (Materials and
Methods). This strain was transformed with pKB5236, containing the
cflB gene and pyrG+ (Materials and Methods). All
transformants isolated showed full complementation of the aerial hyphae and
conidiophore defect.
|
In addition to the null allele cflB, dominant-negative
cflBD123A and dominant-activated
cflBG18V mutant alleles were generated. The D123A mutation
is located in a conserved position that, when mutated in other small GTPases,
results in a dominant-negative phenotype, whereas the G18V mutation in small
GTPases (including RAC homologs) gives rise to a dominant-activated
phenotype because the mutant protein is unable to hydrolyse GTP and
constitutively interacts with downstream effectors
(Davis et al., 1998
;
Fanto et al., 2000
; Kawaski et
al., 1999; Kost et al., 1999
).
Both mutant alleles, as well as a wild-type allele were placed behind the
ethanol-inducible alcA promoter from A. nidulans
(May, 1987
). The
alcA(p)::cflB (pKB5021), alcA(p)::cflBD123A
(pKB5266) and alcA(p)::cflBG18V (pKB5263) constructs were
transformed into strain SPM4 and four representative pyrG+
transformants for each cflB construct were analysed further. The copy
number of these strains ranged from three to eight for alcA(p)::cflB,
from one to ten for alcA(p)::cflBD123A and from two to
three for alcA(p)::cflBG18V. No phenotypic effects caused
by copy number were evident in these transformants. Overexpression of cflB
(alcA(p)::cflB) had no detectable phenotype
(Fig. 3B). By contrast, the
dominant-negative alcA(p)::cflBD123A strains showed a
slightly fluffy phenotype under inducing conditions, with conidiophores that
were reduced in height (Fig.
3B). This phenotype was similar to the
cflB
strain, albeit less severe. The dominant-activated
alcA(p)::cflBG18V strains showed non-uniform conidiation
across the colony on inducing medium (Fig.
3B). Under non-inducing conditions, all strains had a wild-type
phenotype.
cflB is required for asexual development in P.
marneffei
The differentiation of hyphal cells during asexual development requires
regulated changes in polarized growth. P. marneffei initiates asexual
development with the production of a specialized cell type (stalk) from a
vegetative hyphal cell. Sequential budding at the apical end of the stalk
produces sterigmata (metulae and phialides). The sporogenic phialide cells bud
at the apical tip to produce conidia
(Borneman et al., 2000). The
conidiation defects evident in the
cflB,
alcA(p)::cflBD123A and alcA(p)::cflBG18V
strains at the colonial level (Fig.
3) were analysed further microscopically. At 25°C, after 4
days, although conidia were present in the
cflB strain,
conidiophores were not easily distinguishable because the metulae and
phialides were swollen and misshapen, and were occasionally multinucleate
(Fig. 4).
|
Under inducing conditions, the dominant-negative alcA(p)::cflBD123A strains displayed conidiophores with a single, terminal, swollen, multinucleate conidium (Fig. 4). Occasionally, branched conidiophores were also observed (not shown). The dominant-activated alcA(p)::cflBG18V strains also displayed conidiophores with a single, swollen, multinucleate conidium (Fig. 4). However, unlike the alcA(p)::cflBD123A strains, branched conidiophores were not observed.
Correct cflB function is required for asexual development in
A. nidulans
To determine whether the function of CflB is conserved in other fungi, the
alcA(p)::cflB (pKB5021), alcA(p)::cflBD123A
(pKB5266) and alcA(p)::cflBG18V (pKB5263) constructs were
transformed into an A. nidulans pyrG- strain.
Transformants carrying any of these cflB alleles exhibited a severe
decrease in conidiation under inducing conditions and no detectable change in
phenotype under non-inducing conditions
(Fig. 5). In all of the A.
nidulans cflB transformants, conidiophores displayed inappropriate
polarized growth such that metulae and phialides could be greatly extended in
length and were often multinucleate (Fig.
5). Although these extended cells were multinucleate, they were
not septate and failed to conidiate. These strains produced conidia
exclusively from conidiophores with wild-type morphology. In addition to the
conidiation phenotype, the A. nidulans alcA(p)::cflBD123A
transformants also exhibited polarization defects in the vegetative hyphae.
Hyphal cells were occasionally swollen, consequently appearing reduced in
length, and apical cells could be multibranched (data not shown).
|
Deletion of cflB affects germination and results in
misshapen conidia
Wild-type P. marneffei conidia are activated and begin isotropic
growth after 6 hours at 25°C under appropriate growth conditions. After 8
hours, the conidia become highly polarized and, after 12 hours, a germ tube
emerges. Growth at this stage is directed exclusively to the germ-tube apex.
Secondary germ tubes emerge from the conidium after 15 hours incubation
at 25°C. Conidia of the
cflB strain were incubated for 12
hours or 15 hours at 25°C, and germ-tube emergence was measured by
counting the number of germlings with primary, secondary or tertiary germ
tubes in a population of 100 over three independent experiments. After 12
hours, wild-type P. marneffei had 78.3% (±0.88) conidia with
primary germ tubes, 20% (±0.58) conidia with secondary germ tubes and
1.67% (±0.67) conidia with tertiary germ tubes. By contrast,
cflB conidia exhibited an increased rate of secondary
germ-tube emergence with 70.3% (±2.02) primary germ tubes and 26.3%
(±0.88) secondary germ tubes. The proportion of
cflB
conidia with a tertiary germ tube was 3.33% (±1.86), which does not
differ from the wildtype.
After 15 hours at 25°C, 99.7% (±0.33) of wild-type conidia had
germinated (polarized, with one or more germ tubes) and only 0.33%
(±0.33) remain ungerminated. By contrast, the cflB
strain had 20.3% (±1.33) conidia that remained ungerminated after 15
hours. In addition, unlike the uniform ellipsoidal shape of wild-type conidia
(ungerminated or isotropically polarized), 37.5% (±2.22) of germinated
cflB conidia were misshapen. The low germination percentage
and abnormal morphology could be attributed either to defects during
conidiophore development or germination.
CflB controls polarized growth of vegetative cells at 25°C
At 25°C, the SPM4 control strain produced hyphae by the highly
polarized growth of apical cell tips, followed by septation and by the
emergence of new growth tips in subapical cells. Apical cells were never
branched and extended exclusively by polarized growth at the apical tip and
septation occurred at regular intervals. After 2 days growth at 25°C, the
cflB strain displayed apical branching and often showed
multiple branches extending from a single apical cell
(Fig. 6). In the wildtype,
subapical hyphal cells were
40±5 µm in length and produce a
single branch. By contrast, the
cflB mutant extended multiple
branches from subapical cells and these branches were also often branched.
Under inducing conditions, the dominant-negative
alcA(p)::cflBD123A transformants also showed branched
apical hyphal cells and hyperbranching of subapical hyphal cells
(Fig. 6). By contrast, the
alcA(p)::cflB and alcA(p)::cflBG18V strains
showed wildtype branching patterns under inducing conditions. In the wildtype,
nuclei were evenly distributed along a hypha, with older subapical hyphal
cells containing a single nucleus and apical cells containing multiple nuclei.
The nuclear distribution of all of the cflB mutant strains did not
differ from the wildtype after 2 days.
|
After 4 days of growth, the older cells (subapical) showed a reduction in
hyphal cell length to 20±2 µm. The
cflB strain
showed aberrant swollen subapical cells that appeared to be severely reduced
in length (5±2 µm) (Fig.
7). Subapical cells did not exhibit polarized growth, except
during the initiation of new branches. Thus, it appears that the deletion of
cflB results in either inappropriate growth or disruption of cell
integrity, which leads to cell swelling. Staining of cell walls using
calcofluor showed septal positioning defects. Septa were in close proximity,
orientated along random planes rather than perpendicular to the long axis of
the cell and greatly thickened compared to the wildtype. The nuclear index of
these cells varied from none to many (>10 nuclei). Many apical and
subapical hyphal cells were hyperbranched and swelling in hyphal cells varied
between cells (Fig. 7). Under
inducing conditions, the alcA(p)::cflBD123A
dominant-negative transformants also displayed swollen hyphal cells
(10±5 µm), although to a lesser extent than the
cflB
strain, with fewer anucleate or multinucleate subapical cells and
hyperbranched subapical and apical cells
(Fig. 7). The
alcA(p)::cflBG18V transformants showed misshapen swollen
hyphae of varied length (15±10 µm) and nuclear index
(Fig. 7). By contrast, the
alcA(p)::cflB strain showed a wild-type phenotype.
|
Deletion of cflB does not affect yeast cell production
P. marneffei is dimorphic, growing in a filamentous multinucleate
hyphal form at 25°C or in a uninucleate yeast form at 37°C. The
initial stage of conidial germination and polarized growth in P.
marneffei does not appear to differ significantly at either 25°C or
37°C. Conidia expand isotropically and then become highly polarized to
produce a hypha that grows by apical extension and divides by septation.
However, after 4 days at 25°C, the hyphae begin to develop asexual
structures, whereas, after 4 days at 37°C, P. marneffei undergoes
arthroconidiation, which results in the production of rod-shaped yeast cells
(arthroconidia). Double septa are laid down in hyphae and fragmentation occurs
along the septal plane to generate single cells
(Chan and Chow, 1990). The
liberated yeast cells are uninucleate,
20±2 µm in length and
divide by fission (Chan and Chow,
1990
).
After 4 days at 37°C, wild-type P. marneffei yeast colonies
possess uniform edges (Fig.
8A). When the cflB strain was grown for 4 days at
37°C, colonies were greatly reduced in size and displayed irregular edges
(Fig. 8A). When analysed
microscopically, the arthroconidiating hyphae of the
cflB
strain appeared larger in diameter than the wildtype
(Fig. 8B, DIC panel). In
addition, the arthroconidiating hyphae of the
cflB strain
possessed more nuclei per cell and were highly septate
(Fig. 8B, CAL panel). Compared
with the wildtype, arthroconidiation appeared more complete in the
cflB strain, with few true hyphae evident. Despite the
aberrations in cell morphology of the
cflB arthroconidiating
hyphae, normal yeast cells were produced
(Fig. 8C).
|
CflB is required for actin localization during vegetative growth and
asexual development
The cell-polarity defects evident in cflB mutant strain
might be the result of disruption in the actin cytoskeleton because Rac
proteins have been implicated in actin-mediated morphogenesis in several
organisms. We examined the effect of loss of cflB on actin
organization in the various cell types of P. marneffei using a mouse
anti-actin antibody (see Materials and Methods). During hyphal growth of
wild-type P. marneffei at 25°C, actin was localized in cortical
spots along the length of hyphae and concentrated at actively growing branches
and hyphal tips (Fig. 9A).
Actin was present at the nascent septation site before the cell wall was
visible (detected by the calcofluor cell-wall stain). The amount of actin
observed decreased as the septum wall was laid down and mature septa did not
appear to contain actin (Fig.
9C). During asexual development, actin was concentrated at sites
of active cellular morphogenesis and division at the distal tips of stalks,
metulae and phialides, and the nascent conidium bud sites
(Fig. 9B). Cortical actin and
actin at sites of septation were evident in the
cflB strain,
although concentrated actin patches at the hyphal tips were absent
(Fig. 9A,C). Thus, CflB is
required for actin localization at the hyphal tip but not for localization of
cortical spots and actin at nascent septation sites. During asexual
development, the
cflB strain produced conidiophores that were
swollen and aberrant in all sterigmata cells. Actin in conidiophore cells of
the
cflB strain was not localized in developing metulae,
phialides or conidia, suggesting correct actin localization by cflB
is required for morphogenesis of these developmental cell types
(Fig. 9B).
|
During growth of wild-type P. marneffei at 37°C, actin
localized in cortical spots along the arthroconidiating hyphae and was
concentrated at the hyphal tip (Fig.
8B). Similar to what was observed at 25°C, actin formed
structures at nascent septation sites and these were reduced as the cell wall
was laid down and were absent in mature septa. At some septation sites, a
second actin structure was formed as the first reduced and the first septal
wall was laid down. Subsequently, this second actin structure disappeared as
the second septal wall was laid down (Fig.
8D). Yeast cells that had separated from the arthroconidiating
hyphae showed concentrated actin at one end of the cell
(Fig. 8C). Despite the
cflB strain possessing swollen arthroconidiating hyphae with
increased septation, actin was still correctly localized in yeast cells
(Fig. 8B-D).
![]() |
Discussion |
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It is currently unknown whether Rac proteins are involved in cellular
division. Cdc42 in S. cerevisiae and S. pombe has been shown
to be crucial for cytokinesis (Johnson,
1999). If Rac proteins were also involved in cytokinesis, it would
be expected that the protein be localized at division sites. We observed that,
in P. marneffei, CflB was localized at septation sites suggesting
that Rac homologs like Cdc42 have a functional role during cellular division.
This was also supported by the phenotype of the
cflB mutant,
which displayed severe septal positioning defects. It is unclear whether the
septal positioning defects are due to inappropriate cleavage of previously
established compartments or inappropriate spacing when septa are first
deposited. In S. cerevisiae, Cdc42p is localized to the mother-bud
junction and, in S. pombe, Cdc42p is clustered at the site of cell
division (Merla and Johnson,
2000
; Richman et al.,
1999
). In the ectomycorrhizal hyphae of S. bovinus, Cdc42
is evident as two opposing spots at the plasma membrane preceding actin
formation at the nascent septation site and this suggests that Cdc42 in S.
bovinus acts early during cellular division before the formation of the
actin ring (Gorfer, 2001). By contrast, we found that CflB in P.
marneffei is colocalized as rings with actin at nascent septation sites,
suggesting that Rac proteins play a role later in division.
Because Rac proteins are involved in polarized growth, CflB was expected to
be concentrated at sites of polarized growth such as the hyphal tip. This was
not evident in live cells but was detected by immunocytochemistry, presumably
because of the higher sensitivity of this method. In S. cerevisiae,
Cdc42p is clustered at sites of polarized growth, such as incipient bud sites
and the tips and sides of enlarging buds, and this site changes depending on
the stage of the cell cycle (Richman et
al., 2002; Ziman, 1993). Cdc42p in S. pombe was not found
to be concentrated at the cell tips (Merla
and Johnson, 2000
). The CflB localization data suggest that CflB
plays a role during polarized growth. Loss of correct CflB function resulted
in a loss of polarized growth of vegetative hyphae, with hyphae becoming
swollen and misshapen and a loss of concentrated actin at the hyphal tip.
Therefore, CflB acts to maintain polarized growth by directly or indirectly
localizing actin at the growth site. In addition, both the deletion and
dominant-negative strains displayed hyperbranching of apical and subapical
hyphal cells. These data suggest that hyperbranching occurs because of the
lack of correctly focused actin at active apical cells. In the absence of
concentrated actin, growth is not directed and so cells grow in multiple
directions. Alternatively, CflB might negatively control proteins required to
initiate a new site of polarized growth, and loss of CflB function results in
constitutive activity of these factors. This suggests that another protein
takes over after the CflB polarization signal is received, which is consistent
with the phenotype of the dominant-activated transformants, which have
branching patterns similar to the wildtype.
Asexual conidiation is a developmental process that requires, with the
exception of the stalk, the sequential budding of apical cells
(Borneman et al., 2000).
Budding requires the regulated switching on and off of polarized growth in
synchrony with the nuclear division cycle, and the dynamic reorganization of
the actin cytoskeleton. It is clear that CflB, in addition to regulating
polarized growth of vegetative hyphae, also plays a role in regulating
polarized growth during asexual development. All mutant cflB strains
showed conidiation defects and in particular, conidiophores of the
cflB strain were swollen, aberrantly shaped and lacked
localized actin. These results suggest that CflB regulates polarized growth
during asexual development by regulating actin organization. The
dominant-negative transformants, in which the mutant protein is locked in the
GDP-bound inactive form, have conidiophores with reduced length and complexity
that have failed to undergo correct polarized growth, supporting the
suggestion that CflB plays a positive role during polarized growth.
Interestingly, reduced conidiophores are also observed in the P.
marneffei dominant-activated transformants. If CflB acts to turn on
polarized growth, it might be expected that the dominant-activated
transformants would have continuous polarized growth, owing to the inability
to convert the mutant protein into the GDP-bound inactive form. Therefore, not
only must polarized growth be turned on by active GTP-bound CflB but the CflB
protein must be able to cycle appropriately between a GDP-bound and a
GTP-bound form.
Rac proteins have been shown to positively regulate polarized growth in
other organisms. When the Y. lipolytica RAC homolog is deleted, an
impairment of polarized growth in hyphal cells is seen
(Hurtado et al., 2000). It has
also been observed that correct cycling between the GDP- and GTP-bound forms
is essential for positively regulating polarized growth. The expression of the
dominant-negative RAC alleles in the nervous system of D.
melanogaster resulted in occasional loss of axons between the dorsal and
lateral clusters of the peripheral nervous system. Expression of the
dominant-activated allele resulted in a more severe phenotype, with axons
missing between the dorsal and lateral clusters in most segments, in addition
to loss of axons connecting the lateral and ventral clusters
(Luo et al., 1996
). Therefore,
the general function of Rac proteins is to positively regulate polarized
growth by cycling between an inactive and active form, and this is conserved
between distantly related organisms.
We were interested in determining whether Rac proteins function in the same
manner in the analogous process in a closely related organism. The process of
asexual development in A. nidulans is similar to P.
marneffei. In both fungal species, a stalk is produced from the
vegetative hyphae but, in A. nidulans, this stalk swells to produce
the vesicle. Both organisms then undergo sequential budding of cells from the
stalk or vesicle to produce the sterigmata and conidia
(Timberlake, 1987;
Borneman et al., 2000
). In
A. nidulans, overexpression of the wild-type and mutant cflB
alleles resulted in conidiophores that showed hyperpolarized growth. This
occurred in both metulae and phialides, and resulted in multinucleate cells
that remained aseptate. This suggests that, in contrast to P. marneffei,
A. nidulans Rac might function to turn off polarized growth during
asexual development. The difference in the regulation of polarized growth
during asexual development in A. nidulans and P. marneffei
also extends to other Rho GTPases. A. nidulans transformants
expressing mutant cflB alleles show hyperpolarization of
conidiophores, whereas overexpression of the P. marneffei CDC42
homolog cflA in A. nidulans completely blocks conidiation
(Boyce, 2001). In P. marneffei, expression of dominant-negative or
-activated cflB alleles reduces polarized growth during conidiation
but, when the dominant-negative or -activated cflA alleles are
expressed, conidiation is not affected. This highlights the fact that,
although small GTPases are highly conserved in structure and ability to
regulate actin, they can regulate processes differently even in closely
related organisms. It is interesting that the yeasts S. cerevisiae
and S. pombe, and filamentous fungus A. gossypii do not have
Rac orthologs
(http://genome-ftp.stanford.edu/pub/yeast/;
http://nucleus.cshl.edu/pombeweb/;
P. Philippsen, personal communication). The evolution of complicated
developmental programs in higher eukaryotes might have required the evolution
of two proteins that perform similar yet specialized functions. In support of
this, the dimorphic yeasts Y. lipolytica and Candida
albicans, and the complex filamentous fungi A. fumigatus and
P. marneffei do possess Rac orthologs and these organisms show a
larger repertoire of morphogenetic events
(Hurtado et al., 2000
;
Alexopoulos et al., 1996
). In
the case of C. albicans, the Rac has a non-conserved TKVD sequence
(http://www-sequence.stanford.edu/group/candida).
How small GTPases coordinately regulate different aspects of development is
of continuing interest. In P. marneffei, the Cdc42 and Rac proteins
possess coordinate roles during development. Both CflA and CflB are required
for vegetative growth at 25°C, although the roles appear to be distinct
because the phenotypes are not identical
(Boyce et al., 2001). CflA and
CflB also possess distinct roles during development. CflA is required for
yeast cell morphology at 37°C but is not required during asexual
development at 25°C (Boyce et al.,
2001
). By contrast, CflB is not required for yeast cell
morphogenesis but is required during asexual development. In other organisms,
Rho GTPases also possess distinct roles in morphogenesis. For example, the
expression of the dominant activated allele of DmRAC1 during
nervous-system development resulted in stalling of axons, but dendrites were
unaffected. When the dominant-activated CDC42 allele was expressed,
axons were missing and incorrectly positioned, and, in addition, dendrites
were abnormal or absent (Luo et al.,
1994
). Understanding the molecular basis for this coordination
will greatly enhance our understanding of how actin organization and
morphogenesis are regulated throughout development.
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Acknowledgments |
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