Download A role for the DNA-damage checkpoint kinase Chk1 in the virulence

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Research Article
A role for the DNA-damage checkpoint kinase Chk1 in
the virulence program of the fungus Ustilago maydis
Natalia Mielnichuk, Cecilia Sgarlata and José Pérez-Martín*
Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco-UAM, 28049 Madrid, Spain
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 10 September 2009
Journal of Cell Science 122, 4130-4140 Published by The Company of Biologists 2009
doi:10.1242/jcs.052233
Summary
During induction of the virulence program in the
phytopathogenic fungus Ustilago maydis, the cell cycle is
arrested on the plant surface and it is not resumed until the
fungus enters the plant. The mechanism of this cell cycle arrest
is unknown, but it is thought that it is necessary for the correct
implementation of the virulence program. Here, we show that
this arrest takes place in the G2 phase, as a result of an increase
in the inhibitory phosphorylation of the catalytic subunit of the
mitotic cyclin-dependent kinase Cdk1. Sequestration in the
cytoplasm of the Cdc25 phosphatase seems to be one of the
reasons for the increase in inhibitory phosphorylation.
Introduction
Developmental decisions often involve differentiation processes that
need to reset the cell cycle for induction of a new morphogenetic
program. This is probably also the case for induction of the virulence
program in pathogenic fungi. It could therefore be assumed that in
pathogenic fungi the control of the cell cycle, as well as
morphogenesis, is linked somehow to the virulence program. The
maize smut fungus Ustilago maydis is an excellent system to address
the relationships between cell cycle, morphogenesis and
pathogenicity (Perez-Martin et al., 2006; Steinberg and PerezMartin, 2008). During the induction of virulence program in this
fungus, an infectious dikaryotic hypha is produced as a result of
the mating of a pair of compatible haploid budding cells. Therefore,
the induction of the pathogenic program implies not only strong
morphological changes (bud to hypha transition) but also genetic
changes (haploid to dikaryotic transition). Accurate control of the
cell cycle and morphogenesis is predicted during these transitions.
The formation of the infectious hypha in U. maydis depends on
an intricate transcriptional program which primarily involves a
transcriptional regulator called b-factor (Feldbrugge et al., 2004).
The production of this master regulator is linked to the mating
process that, after cell fusion, leads to the interaction of the two
subunits composing the b-factor (bW and bE), one provided by each
mating partner. This way, the mating of two compatible cells (i.e.
carrying b-subunits able to dimerize) results in the formation of a
dikaryotic cell, which undergoes a strong polar growth as well as
an apparent cell cycle arrest on the plant surface. Eventually, the
infectious hypha manages to enter the plant tissue, where it starts
to proliferate, maintaining its dikaryotic status. The reasons for the
apparent cell cycle arrest on the plant surface are unknown. It has
been hypothesized that such a cell cycle adjustment would be
required for a precise execution of the virulence program (PerezMartin et al., 2006). In fact, the apparent cell cycle arrest of the
infectious hypha on the plant surface observed in U. maydis seems
Strikingly, we also report the DNA-damage checkpoint kinase
Chk1 appears to be involved in this process. Our results
support the emerging idea that checkpoint kinases have roles
other than in the DNA-damage response, by virtue of their
ability to interact with the cell cycle machinery.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/22/4130/DC1
Key words: Cell cycle, Ustilago maydis, Chk1, Phytopathogenic
fungi
to be more general, and it is also present in rust fungi such as
Uromyces phaseoli (Heath and Heath, 1979).
Ectopic expression of compatible b-subunits in a haploid cell
induces the formation of a monokaryotic filament that mimics its
dikaryotic counterpart in all aspects of filamentous growth as well
as apparent cell cycle arrest (Brachmann et al., 2001). Which
mechanisms are used by the bW-bE heterodimer to induce the
hyperpolarized growth and to arrest the cell cycle in the infectious
hypha and at which stage this occurs are unknown, but these issues
are currently being studied. For example, recent results from our
laboratory indicated that the activation of hyperpolarized growth
after bW-bE heterodimer formation is dependent on the U. maydis
ortholog of the cyclin-dependent kinase Cdk5, a well-known
regulator of neuron development in mammals (Alvarez-Tabares and
Perez-Martin, 2008; Castillo-Lluva et al., 2007; Flor-Parra et al.,
2007).
Here, we address some of the questions related to the b-factorinduced cell cycle arrest, showing that such cell cycle arrest takes
place in the G2 phase as a consequence of an increase of inhibitory
phosphorylation of the mitotic cyclin-dependent kinase.
Interestingly, we also report that the DNA-damage checkpoint
kinase Chk1 seems to be involved in this process.
Results
G2 cell cycle arrest in U. maydis upon expression of
compatible b-homeoproteins
To address the molecular mechanisms of the b-factor-dependent cell
cycle arrest, we made use of the previously described (Brachmann
et al., 2001) haploid U. maydis AB33 strain, which carry compatible
(i.e. able to dimerize) bE and bW genes under the control of the
regulatable nar1 promoter. As the nar1 promoter can be induced
by growing cells in nitrate-containing medium (MM-NO3) and
repressed in ammonium-containing medium (MM-NH4), b-factordependent infective filament formation can be elicited in AB33 by
Journal of Cell Science
Virulence-induced G2 arrest in smuts
changing the nitrogen source. As a control we used a similar strain,
AB34, which harbors incompatible bE and bW genes (i.e. unable
to produce dimers) under the control of nar1 promoter (Brachmann
et al., 2001) (Fig. 1A).
We measured the DNA content of these strains under induction
and non-induction conditions using fluorescence-activated cell
sorting (FACS) analysis and found that cells expressing compatible
b-proteins accumulated with a 2C DNA content (Fig. 1B). To
specifically ascribe the cell cycle stage at which arrest takes place,
we introduced two cytological markers that helped us to distinguish
G2 from early mitosis in a strain expressing compatible b-proteins:
an -tubulin-GFP protein fusion allows the detection of the
microtubule cytoskeleton (Steinberg et al., 2001) and a Cut11-RFP
fusion protein labels the nuclear envelope (Pérez-Martín, 2009).
During the G2 phase, the nuclear envelope is present and the
microtubules form a cytoplasmic network (Fig. 1C, inset arrow);
however, once cells enter mitosis, the nuclear envelope is
disassembled and the microtubules are concentrated in the spindle
(Fig. 1C, inset, arrowhead) (Steinberg et al., 2001; Straube et al.,
2005). Filaments produced after expression of compatible bproteins showed a single nucleus surrounded by an intact nuclear
envelope and a defined cytoplasmic array of microtubules (Fig. 1C,
arrows), all of which, together with the 2C DNA content, is
consistent with a G2 cell cycle arrest during the formation of the
infective filament.
G2 cell cycle arrest is dependent on the inhibitory
phosphorylation of Cdk1
G2-M transition in U. maydis is enabled by the activity of two CDK
complexes: Cdk1-Clb1 and Cdk1-Clb2 (Garcia-Muse et al., 2004;
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Sgarlata and Perez-Martin, 2005a). To determine the kinase activity
associated with Cdk1 during b-factor-dependent filament formation,
we took advantage of the Schizosaccharomyces pombe protein Suc1,
which is known to bind specifically to mitotic CDKs with high
affinity (Ducommun and Beach, 1990) and that we previously
proved to be a convenient assay for characterizing the U. maydis
Cdk1 protein (Garcia-Muse et al., 2004). Cell lysates obtained from
AB33 and AB34 strains growing in induction (MM-NO3) and noninduction (MM-NH4) conditions were incubated with Suc1conjugated Sepharose beads and purified fractions assayed for
histone H1 kinase activity (Fig. 2A). Consistently with the inability
of the hyphae to enter mitosis, Cdk1-associated kinase activity was
downregulated by more than 90% after induction of the expression
of compatible b-factor genes (Fig. 2B).
Downregulation of CDK activity can be produced in different
ways in U. maydis (Perez-Martin et al., 2006), one of these, which
is particularly active during the control of G2-M transition, is the
phosphorylation of the Tyr15 residue in Cdk1 (Sgarlata and PerezMartin, 2005a). Therefore, we analyzed levels of Cdk1 inhibitory
phosphorylation upon induction of b-factor genes. For this, we used
a specific antibody raised against the phosphorylated human Cdc2Y15-P peptide, which recognizes the Tyr15-phosphorylated form
of U. maydis Cdk1 (Sgarlata and Perez-Martin, 2005a). Tyr15P-Cdk1
levels increased eightfold when compatible b-proteins were
expressed (Fig. 2C,D).
To address the importance of this phosphorylation during the bfactor-induced cell cycle arrest, we took advantage of the cdk1AF
allele, in which the inhibitory phosphorylation sites in Cdk1 are
replaced with residues that cannot be phosphorylated (Thr14 to Ala
and Tyr15 to Phe, respectively) (Sgarlata and Perez-Martin, 2005a).
Fig. 1. Expression of b-homeoproteins in Ustilago maydis produces a G2 cell cycle arrest. (A)Scheme of the cassettes expressing compatible (AB33) or noncompatible (AB34) b-factor genes. Only the compatible pair is able to form the heterodimer. (B)FACS analysis of the DNA content of AB34 and AB33 strains
growing in inducing (MM-NO3) and non-inducing (MM-NH4) conditions. The period of incubation in testing medium is indicated (hours). (C)Cell images of
AB33-derived cells carrying Tub1-GFP and Cut11-RFP fusions to detect the microtubule cytoskeleton (arrows) and the nuclear envelope growing in conditions that
induce the expression of the b-factor genes (MM-NO3). Different stages during the production of the infective hyphae are shown. The inset shows the same strain
growing in non-inducing conditions (MM-NH4). Two cells are shown, one in G2 phase (the nuclear envelope is present and the microtubules are forming a
cytoplasmic network, arrow) and the other is in the middle of mitosis (there is no nuclear envelope signal and the microtubules are concentrated in the spindle,
arrowhead). Scale bars: 10m.
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Fig. 2. b-factor-induced Cdk1 inactivation in U. maydis.
(A)Kinase activity of the Cdk1 complexes purified using
Suc1 beads obtained from AB33 and AB34 cells growing
in inducing and non-inducing conditions for 8 hours. The
upper panel shows an immunoblot using anti-PSTAIRE
antibody, which detects Cdk1 and Cdk5 kinases in U.
maydis; only Cdk1 binds to Suc1 beads. The Suc1 protein
precipitates were used for in vitro phosphorylation of
histone H1 (lower panel, autoradiograph of SDS-PAGE).
(B)Quantification of Cdk1 kinase activity after b-factor
expression. Kinase activity obtained from AB34 cells
growing in non-induction conditions (MM-NH4) was used
as reference (100% of activity). Means ± s.d. are shown.
(C)Levels of Cdk1 inhibitory phosphorylation after bfactor induction. Protein extracts from the indicated
strains growing in inducing conditions (MM-NO3) for the
indicated time (hours) were separated by SDS-PAGE.
Immunoblots were incubated successively with an
antibody that recognizes the Cdk1 phosphorylated form
(Cdc2-Y15P) and anti-PSTAIRE. (D)Levels of Cdk1
phosphorylation were determined by quantifying the level
of antibody signal using ChemiDoc (Bio-Rad). Signal
from the phosphopeptide-specific antibodies was
normalized to the amount of phosphorylation of control
strain (AB34) at time zero. Differences in loading of
samples were corrected by dividing each phosphopeptidespecific antibody signal by the Cdk1 (PSTAIRE) antibody
signal. Means ± s.d. are shown.
As continuous expression of this allele produces a deleterious effect
in the cell (Sgarlata and Perez-Martin, 2005a), we decided to
introduce an ectopic copy of this mutant allele under the same
promoter as the b-factor genes (i.e. nar1 promoter) in AB33 cells,
in such a way that production of Cdk1AF occurs at the same time as
the cell produces b-proteins. As a control, we constructed an AB33
strain carrying an ectopic copy of a wild-type cdk1 allele under control
of the nar1 promoter. We used 3⫻Myc-tagged versions of both the
wild-type and mutant ectopic Cdk1 to discriminate between the levels
of protein produced by the endogenous locus and the ectopically
expressed alleles (Fig. 3A). We found that in the strain expressing
the Cdk1 version refractory to inhibitory phosphorylation (AB33
cdk1AF), the Cdk1-associated kinase activity remained high. By
contrast, in AB33 or in the control strain expressing a Cdk1 version
that can be phosphorylated (AB33cdk1), the kinase activity was
downregulated after b-factor expression (Fig. 3B). Consistently, in
the presence of Cdk1AF mutant protein, the b-factor-dependent
filaments carried several nuclei, indicating that no cell cycle arrest
Fig. 3. Cdk1 inhibitory phosphorylation is required for bfactor-induced cell cycle arrest in U. maydis. (A)Strains
AB33 Pnar1:cdk1-myc and AB33 Pnar1:cdk1AF-myc, in
addition to the endogenous cdk1 wild-type allele, carry an
ectopic extra copy of a cdk1-myc or cdk1AF-myc allele
under the control of nar1 promoter, in such a way that
conditions inducing b-factor gene expression also induced
the production of the Myc-tagged Cdk1 proteins. Panel
shows western blot using anti-PSTAIRE antibody of the
different strains, after 8 hours in inducing conditions
(MM-NO3). Note the levels of Cdk-Myc and Cdk1AF-Myc
proteins with respect to endogenous levels of wild-type
Cdk1. (B)Kinase activity of the indicated strains. Activity
of the control strain AB34 growing in MM-NO3 was used
as reference (100% activity). (C,D)Cell images of AB33
(control) and AB33-derived strains expressing cdk1-myc
(cdk1) or the non-phosphorylatable cdk1AF-myc (cdk1AF)
alleles, incubated for 8 (C) and 24 (D) hours in inducing
conditions (MM-NO3). Cells were stained with DAPI to
detect nuclei. Scale bars: 20m.
Virulence-induced G2 arrest in smuts
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was taking place (Fig. 3C,D). Expression of wild-type Cdk1 produced
filaments that were indistinguishable from those obtained with the
AB33 control strain. Taken together, these results indicate that
inhibitory phosphorylation of Cdk1 has a major role during the bfactor-induced G2 arrest in U. maydis.
Journal of Cell Science
Cdk1 inhibitory phosphorylation during b-factor-induced cell
cycle arrest depends on Wee1
Cdk1 inhibitory phosphorylation during vegetative growth depends
on the activity of the essential Wee1 kinase (Sgarlata and PerezMartin, 2005a). To address whether Wee1 is required for b-factorinduced cell cycle arrest, we took advantage of previously described
strain (AB31) (Brachmann et al., 2001) that expresses compatible
b-factor genes under control of the crg1 promoter, which is induced
in growth medium containing arabinose as a carbon source and
repressed when glucose is present (Bottin et al., 1996). We
introduced the conditional allele wee1nar1 in this strain (Fig. 4A)
(Sgarlata and Perez-Martin, 2005a). This allele enables the
expression of wee1 in cells incubated in minimal medium plus
nitrate as a nitrogen source (MM-NO3) as well as the downregulation
of wee1 expression in complete medium (CM, Fig. 4B).
When AB31-derived wee1 conditional cells were grown in
repressive conditions (CM) and in the presence of arabinose,
multinucleated filaments were observed (Fig. 4C). In similar
conditions, AB31 cells had a single nucleus, indicating that Wee1
is necessary for b-factor-induced cell cycle arrest. Consistently, the
inability to arrest the cell cycle in the absence of Wee1 correlated
with a dramatic decrease in the level of Tyr15-P Cdk1, as well as
a high level of Cdk1-associated kinase activity (Fig. 4D,E).
b-factor-dependent cell cycle arrest is not solely mediated by
transcriptional regulation of wee1
We analyzed the levels of wee1 mRNA in AB33 cells after b-factor
induction and found a sevenfold increase with respect to the AB34
control strain (Fig. 5A and supplementary material Fig. S1). Since
overexpression of wee1 produces a G2 cell cycle arrest (Sgarlata
and Perez-Martin, 2005a), we wondered whether this upregulation
was responsible for b-factor-induced cell cycle arrest. To answer
this question, we exchanged the endogenous wee1 native promoter
with the constitutively expressed scp promoter in AB33 cells (Fig.
5B). This mutant allele, wee1scp, produces a low and constitutive
level of wee1 mRNA without any appreciable defect in cell cycle
regulation in normal conditions (Sgarlata and Perez-Martin, 2005a).
In AB33 wee1scp cells, wee1 mRNA level was maintained at a low
level upon b-factor induction (Fig. 5C) However, we found a minor
effect in cell cycle arrest: less than 25% of filaments showed more
than one nucleus (Fig. 5D,E), indicating that the observed
transcriptional upregulation of wee1 is not the only reason by which
cell cycle arrests during b-factor induction.
Ectopic cdc25 overexpression impairs the b-factor-induced cell
cycle arrest
The level of inhibitory phosphorylation of Cdk1 also depends on
the activity of the Cdc25 phosphatase, which reverses the CDK
inhibition (Sgarlata and Perez-Martin, 2005b). Therefore, we
analyzed the levels of cdc25 mRNA during b-factor-induced
filamentation. However, we found that it was not affected by the
presence of an active b-heterodimer (Fig. 6A). In spite of this result,
we wondered whether it was possible to release the cell cycle arrest
by overexpressing cdc25. To achieve this, we introduced in a strain
carrying compatible b-factor genes under control of the nar1
Fig. 4. Wee1 is required for b-factor-dependent cell cycle arrest. (A)Scheme
showing the constructions inserted in the strains used. AB31 carries
compatible b-factor genes under the control of the crg1 promoter that can be
induced by arabinose as a carbon source and repressed in the presence of
glucose. In AB31wee1nar1, the endogenous promoter of wee1 was exchanged
with the regulatable nar1 promoter. (B)Levels of wee1 mRNA in the indicated
strains when cells were grown in conditions for b-factor gene expression (1%
arabinose) and conditions for nar1 expression (MM-NO3) or repression (CM,
complete medium carrying casamino acids and NH4 as nitrogen source).
(C)Cell images of AB31 (control) and AB31-derived strain carrying a
conditional wee1nar1 allele (wee1OFF) after 8 and 24 hours of incubation under
conditions of expression of b-factor genes and repression of wee1 (complete
medium plus arabinose). Cells were stained with DAPI to detect nuclei.
(D)Levels of Cdk1 phosphorylation of AB31 and AB31 wee1nar1 cells
growing for the indicated time in complete medium plus arabinose as carbon
source. Quantification was as above. (E)Quantification of Cdk1 kinase
activity after b-factor expression in the indicated strains. Kinase activity
obtained from control AB32 cells growing in induction conditions (MM-NO3)
was used as reference (100% activity). Means ± s.d. are shown.
promoter (AB33 cells), an ectopic copy of cdc25 under the control
of the dik6 promoter, which is strongly induced by the b-proteins
(Brachmann et al., 2001) (Fig. 6B). In the resulting strain, induction
of b-factor genes resulted in a dramatic increase in cdc25 mRNA
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Fig. 5. Transcriptional regulation of wee1 has a minor role during
b-factor-dependent cell cycle arrest. (A)Northern analysis of wee1
mRNA levels in strains carrying compatible (AB33) or
incompatible (AB34) b-factor genes and grown for the indicated
time (hours) in inducing (MM-NO3) or repressing (MM-NH4)
conditions for b-factor expression. Ribosomal RNA was used as
loading control. (B)Scheme showing the genes relevant for the
experiments showed in panels C-E. The scp promoter transcribes
at a low and constitutive level of expression. (C)Levels of wee1
mRNA in the indicated strains when cells were grown in
conditions for b expression (MMNO3) for the indicated time
(hours). (D)Images of the AB33-derived strain carrying the
wee1scp allele incubated for 8 hours in induction conditions. Cells
were stained with DAPI to detect nuclei. (E)Quantification of
filaments carrying single nucleus after 24 hours of incubation.
Mean and s.d. are shown.
levels (Fig. 6C). Interestingly, the filaments expressing high levels
of cdc25 had more than one nucleus in a substantial proportion
(64% of the filaments) (Fig. 6D). Furthermore, the cells expressing
cdc25 under control of the dik6 promoter showed a low level of
inhibitory phosphorylation of Cdk1 (Fig. 6E).
Altogether, the results obtained with Cdc25 and Wee1 indicated
that a similar regulatory scheme controlling G2-M transition via
CDK inhibitory phosphorylation during vegetative growth is also
involved in b-factor-induced cell cycle arrest.
Cdc25 seems to be retained in the cytoplasm during b-factorinduced filamentation
The observation that overexpression of cdc25 resulted in a
multinucleated filament can be explained if b-factor-induced cell
cycle arrest relies on an inhibition of Cdc25. One way to control
Cdc25 activity in U. maydis cells is to regulate its subcellular
localization (Mielnichuk and Perez-Martin, 2008). We explored this
possibility by monitoring Cdc25 nucleus to cytoplasm distribution
in cells expressing GFP-tagged Cdc25 from the genomic locus. To
quantitatively evaluate changes in localization, fluorescence
intensities of the Cdc25 signal were determined for a circular region
corresponding to the nucleus (N) and a comparable area in the
cytoplasm (C), as described (Mielnichuk and Perez-Martin, 2008).
We found that in inductive conditions (MM-NO3) the ratio N/C in
control cells (AB34 cells) was about 1.1, whereas this ratio in AB33
cells was around 0.2 (Fig. 7A).
Previous work from our laboratory showed that retention of
Cdc25 in the cytoplasm of U. maydis depends on the essential
14-3-3 protein Bmh1 (Mielnichuk and Perez-Martin, 2008). To
address whether 14-3-3 was required for b-factor-induced cell cycle
arrest, we expressed compatible b-factor genes in conditions of
downregulation of bmh1 expression (Fig. 7B). We found that in
these conditions, morphologically aberrant filaments were produced
that contained several nuclei each (Fig. 7D, middle panel). Since
14-3-3 proteins are extremely pleiotropic (van Hemert et al., 2001),
it is hard to unequivocally ascribe the effects observed in
filamentation after bmh1 downregulation to the inability to arrest
cell cycle. To link this directly to a defect in the ability to retain
Cdc25 in the cytoplasm, we took advantage of a cdc25 mutant allele
(cdc25AAA, Fig. 7C), which was impaired in the interaction with
Bmh1 and therefore accumulates in the cell nucleus (Mielnichuk
and Perez-Martin, 2008). We found that a strain carrying the
cdc25AAA allele and expressing compatible b-proteins resulted in a
high percentage of filaments carrying more than one nucleus (Fig.
7D, bottom panel). Collectively, these data suggest that custody of
Cdc25 in the cytoplasm is part of the mechanism by which b-factordependent cell cycle arrest occurs.
Chk1 is involved in the b-factor-dependent cell cycle arrest
Cytoplasmic retention of Cdc25 by 14-3-3 proteins requires the
previous phosphorylation of target sites in Cdc25 (van Hemert et
al., 2001). The DNA-damage checkpoint kinase Chk1 is one of the
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Virulence-induced G2 arrest in smuts
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Fig. 6. Expression of cdc25 under control
of the dik6 promoter. (A)Northern analysis
of cdc25 mRNA levels in strains carrying
compatible (AB33) or incompatible
(AB34) b-factor genes and growing for the
indicated time (hours) in inducing (MMNO3) or repressing (MM-NH4) conditions
for b-factor expression. Ribosomal RNA
was used as loading control. (B)Scheme
showing the constructions inserted in the
different strains. AB33 Pdik6:cdc25 carries
an additional ectopic copy of the cdc25
gene under the control of the dik6
promoter that is activated by the presence
of a compatible b-heterodimer.
(C)Northern analysis of cdc25 levels in the
control strain (AB33) and the strain
carrying the ectopic copy of cdc25 under
dik6 control growing in non-inducing
(MM-NH4) and inducing (MM-NO3)
conditions for the time indicated (hours).
(D)Images of an AB33-derived strain
carrying an ectopic copy of cdc25 under
the control of the b-factor-induced dik6
promoter after 8 and 24 hours of growth in
conditions for expression of b-factor genes
(MM-NO3). Cells were stained with DAPI
to detect nuclei. (E)Levels of Cdk1
phosphorylation of AB33 and AB33
Pdik6:cdc25 cells growing for the indicated
time in MM-NO3. Quantification was as
above. Means ± s.d. are shown.
described kinases able to phosphorylate Cdc25 (Karlsson-Rosenthal
and Millar, 2006). Interestingly, the mutations carried in the
cdc25AAA allele affect the ability of the DNA-damage checkpoint
kinase Chk1 to arrest the cell cycle in U. maydis (Perez-Martin,
2009). Therefore, we analyzed whether Chk1 was required for bfactor-induced cell cycle arrest. To our surprise, cells defective in
Chk1 and expressing compatible b-proteins were impaired in their
ability to arrest the cell cycle (Fig. 8A,B).
Even when the b-factor-dependent filament produced in AB33
cells mimics its dikaryotic counterpart in all aspects of filamentous
growth, we sought to analyze the consequences of the absence of
chk1 in the formation of the infective filament in more ‘native’
conditions. Crosses of compatible haploid wild-type cells on
charcoal-containing plates (Banuett and Herskowitz, 1989) resulted
in the formation of cell-cycle-arrested dikaryotic hyphae (Flor-Parra
et al., 2006). Therefore, we crossed compatible wild-type and chk1
strains on charcoal-containing plates. Wild-type crosses led to a
filamentous appearance of the colony, whereas chk1 mutants were
strongly attenuated in filament formation (Fig. 8C,D). We also
analyzed the nuclear composition of filaments in these crosses. To
distinguish the b-factor-induced filaments from the cell population
background (frequently enriched in aberrant elongated cells) the
haploid strains we used expressed a GFP fusion to a nuclear
localization signal under control of the dik6 promoter. In this way,
only cells resulting from mating and therefore expressing the bfactor-dependent program produced a fluorescent nuclear signal.
We found that almost all cells expressing the b-factor carried two
nuclei, whereas chk1 filaments frequently carried more than two
nuclei (Fig. 8E,F), which was consistent with a defect in the ability
to arrest entry into mitosis during the formation of the infective
hyphae.
Chk1 is transiently activated during b-factor-induced cell cycle
arrest
The above results indicated that during induction of the pathogenic
development in U. maydis, a G2 cell cycle arrest takes place in
which the checkpoint kinase Chk1 has a role. Chk1 is a protein that
is produced in normal conditions in an inactive form, which is
activated after phosphorylation by upstream kinases (Chen and
Sanchez, 2004). Therefore, we examined whether Chk1 was
activated during the formation of the b-factor-dependent filament.
In U. maydis cells, as in other organisms, Chk1 activation can be
easily monitored by the accumulation of a GFP-tagged Chk1 protein
into the nucleus (Perez-Martin, 2009). When AB33-derived cells
carrying a Chk1-GFP fusion were induced to produce filaments,
we observed a clear accumulation of the GFP signal in the nucleus.
Control AB34 cells showed no accumulation of GFP in the nucleus
throughout the incubation period (Fig. 9A). Interestingly, AB33derived long filaments seemed not to accumulate any nuclear GFP
signal. To quantify this apparently transient response, we measured
cells (n80 in two independent experiments) of different length and
plotted this against the presence or not of a nuclear GFP signal (Fig.
9B), confirming that only shorter filaments (i.e. early stages) showed
nuclear accumulation of Chk1.
There are several possible explanations for this behavior. One
possibility is that Chk1 is degraded at later stages. A different
explanation is that even when the nuclear membrane is present
during filament formation (see Fig. 1C), it becomes permeable at
later stages. And finally, it is possible that Chk1 is transiently
activated during b-factor-dependent filamentation. To address these
possibilities, we monitored the decrease in the mobility of a Myctagged protein during electrophoresis as a surrogate marker for their
phosphorylation-dependent activation (Perez-Martin, 2009). First,
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Fig. 7. Cytoplasmic retention of Cdc25 is required for b-factor-induced cell cycle arrest in U. maydis. (A)Subcellular localization of Cdc25 in AB34 (noncompatible b-factor genes) and AB33 (compatible b-factor genes) derivate cells carrying an endogenous functional Cdc25-3GFP fusion. Cells were incubated
under inducing conditions (MM-NO3) for 8 hours. Graph indicates the quantification of nucleus to cytoplasm ratio of GFP fluorescence signal. Quantification of
fluorescence signal was performed by measuring pixel intensity in the nuclei and in an equivalent area of the cytoplasm. Mean and s.d. are shown. (B)Scheme
showing the construction carried by the strain that express b proteins under conditions of down-regulation of the 14-3-3 Bmh1 protein. The bmh1crg1 allele is
repressed in the presence of glucose and transcribed when arabinose was the only carbon source. (C)Schematic representation of the cdc25AAA allele showing the
amino acid changes introduced to void the 14-3-3 binding sites. The phosphatase catalytic domain (PPase) and the putative nuclear localization signal (NLS) of
Cdc25 are also showed. (D)Cell images of AB33 (control) and AB33-derived strain carrying either a conditional bmh1crg1 allele (bmh1OFF) or the cdc25AAA allele
that has an impaired interaction with the 14-3-3 protein Bmh1 (cdc25AAA), after 8 hours of incubation in induction conditions for expression of b-factor genes (MMNO3, bmh1crg1 was repressed in these conditions because the carbon source was glucose). Cells were stained with DAPI to detect nuclei.
we observed that the overall levels of Chk1 were not affected by
the incubation period, excluding degradation of Chk1 as a possible
explanation (Fig. 9C). Interestingly, the electrophoretic shift was
maximum after 3 hours of induction but decreased to negligible
after 9 hours, supporting the explanation that Chk1 is transiently
activated during the formation of the b-factor-dependent filament.
were sampled after 1 week of inoculation and Chlorazole Black E
staining was used to visualize invading hyphae (Brachmann et al.,
2003). Septated hyphae grown through epidermal cells were observed
in plants inoculated with compatible wild-type strains. When material
obtained from mutant crosses was analyzed, mutant hyphae were
observed, although they showed a more branched appearance with
shorter cell compartments with lobed tips (Fig. 10B).
Chk1 is required for full virulence
U. maydis infection of maize results in anthocyanin pigment
production by the plant and the formation of tumors that are filled
with proliferating fungal cells, which eventually differentiate into
black teliospores (Banuett and Herskowitz, 1996). To address the
consequences of a defective cell cycle arrest during corn infection
by U. maydis, we inoculated maize plants by stem injection with
mixtures of wild-type and chk1 mutant strains. Both crosses were
able to infect plants; however, the mutant strains were less efficient
(42% of the plants showed no symptoms) and the severity of the
symptoms was minor (Fig. 10A). Interestingly, plants infected with
the chk1 mutant rarely showed large tumors (Fig. 10C), and even
in these rare occasions, no teliospores were found, suggesting a
role of Chk1 beyond the initial steps of infection (i.e. infective
filament formation).
To address whether the mutant hyphae proliferate inside the plant,
symptomatic leaves obtained from both mutant and wild-type crosses
Discussion
Here, we showed that formation of an active bW-bE heterodimer
in Ustilago maydis results in a sustained cell cycle arrest in G2
phase. This cell cycle arrest is a consequence of the accumulation
of phosphorylated inactive forms of Cdk1, the mitotic CDK in this
fungus. Inability to phosphorylate this protein, either by
downregulation of the kinase Wee1, or by expression of a Cdk1
allele that is refractory to inhibitory phosphorylation, resulted in
filaments that were not arrested in the cell cycle. We tried to
determine which molecular mechanisms were responsible for such
accumulation of phosphorylated forms of Cdk1. Taken together,
our results support the idea that upon induction of b-factor genes,
the prevention of nuclear accumulation of Cdc25 by interaction with
14-3-3, which is helped by the upregulation of wee1, results in an
increase in the inhibitory phosphorylation of Cdk1, which inhibits
the G2-M transition during the formation of infective hyphae.
Journal of Cell Science
Virulence-induced G2 arrest in smuts
4137
Fig. 8. Chk1 is required for b-factor-induced cell cycle arrest. (A)Cell images of AB33 (control) and a derivate strain lacking the chk1 gene incubated for 8 hours
in inducing conditions (MM-NO3). Cells were stained with DAPI to detect nuclei. (B)Percentage of cells producing mononucleated filaments (i.e. cell-cycle
arrested) after 24 hours of incubation; means ± s.d. are shown. (C)Scheme of the formation of white filamentous dikaryon. a1b1 cells are indicated in white, a2b2
cells are indicated in dark gray, and the dikaryon is shown in light gray. Filamentous growth is indicated by thin lines rising from the colony. (D)Crosses of control
strains FB1 x FB2 and chk1 mutant strains in charcoal-containing agar plates. Note the gray appearance of mutant cross indicating impairment in filament
formation. (E)Compatible strains carrying a NLS-GFP fusion under control of the b-factor-dependent dik6 promoter were scrapped from agar surface, mounted on
microscopy slides and epifluorescence was observed. Upper image shows DIC images of cells and bottom image shows fluorescence in GFP channel.
(F)Quantification of the nuclear content of filaments obtained from charcoal plates.
A surprising result was the realization that Chk1, a well-known
regulator involved in the DNA-damage response, has roles in this
cell cycle arrest. Furthermore, we found that Chk1 was transiently
activated during formation of the infective hypha. Transient
activation of Chk1 is compatible with the proposed effect on the
cell cycle of these checkpoint systems, which appear to be more
devoted to induce a transient arrest (thus providing time to solve
the problems) rather than a permanent arrest (Toettcher et al., 2009).
It could also be that additional elements are required to sustain the
observed cell cycle arrest, and that Chk1 activation is just the trigger.
It is unclear how the expression of b-factor genes is linked to Chk1
activation. We found no increase in either mRNA or protein levels
of Chk1 upon b-factor induction (not shown). As Chk1 activation
is linked to DNA damage in vegetative cells (Perez-Martin, 2009),
we analyzed whether there was DNA damage associated with the
induction of the infective hyphae by looking for the formation of
Rad51 foci as a reporter (Kojic et al., 2008) (supplementary
material Fig. S2). We found no proof of massive DNA damage (i.e.
no accumulation of Rad51 foci were observed); however, we cannot
discard the idea that limited DNA damage is responsible for Chk1
activation. It is interesting to note that one of the first genes described
in which transcription was directly activated by b-proteins encoded
a putative DNA polymerase-, which is one member of the X family
of DNA polymerases that are involved in a number of DNA repair
processes (Brachmann et al., 2001; Ramadan et al., 2004).
Unfortunately, no role has been determined so far for this factor in
U. maydis.
An interesting question that has not yet been addressed is the
reason for such a cell cycle arrest. We believe that this specific cell
cycle arrest in U. maydis has a mechanistic role. In U. maydis, the
G2 phase is characterized by polar formation of a bud, which
requires the rearrangement of the cytoskeleton and involves
specialized sets of motors, such as cytoplasmic dynein (Steinberg
et al., 2001; Straube et al., 2001), which support the polar extension
of the cell. In other words, in the G2 phase, the cytoskeletal growth
machinery is set up to support polar growth. Assuming that the
Journal of Cell Science
4138
Journal of Cell Science 122 (22)
Fig. 9. Chk1 is transiently activated during b-factor filamentation. (A)Chk1 is
localized transiently in the nucleus during b-factor-dependent filamentation.
Images of an AB33-derived strain carrying a Chk1-3GFP fusion grown in
MM-NO3 for 3 (top right panel), 6 (middle right panel) and 9 hours (bottom
right panel). Control strain AB34 carrying a Chk1-3GFP fusion was grown in
MM-NO3 for 3 hours (left panel). All cells are shown at the same
magnification. (B)Distribution of cells showing nuclear GFP accumulation in
function of cell length. Quantification is the result of measurement of two
independent experiments, counting 80 cells in each. Means ± s.d. are shown.
(C)In vivo phosphorylation of Chk1 during b-factor-dependent filamentation.
AB33- and AB34-derived cells carrying an endogenous Chk1-3⫻Myc allele
were incubated in MM-NO3 for the indicated time (in hours). Protein extracts
were immunoprecipitated with a commercial anti-Myc antibody and
immunoprecipitates were subjected to SDS-PAGE and immunoblotted with
anti-Myc antibody.
formation of the infective hyphae is based on a similar mechanism
as polar bud growth, a prolonged G2 phase is best suited to support
tip growth during this stage.
An additional interesting question that emanates from our results
concerns the additional roles that Chk1 might have during the
pathogenic process, when the fungus is growing inside the plant.
A simple answer would be related to the ability of fungal cells to
deal with DNA damage occurring during the proliferation inside
the plant in response to the plant defense system (i.e. in the form
of reactive oxygen species), because mutants defective in Chk1
function were extremely sensitive to DNA-damage agents (PérezMartín, 2009). However, we believe that this is not the case, because
fungal cells defective in Brh2, the BRCA2 homolog, were able to
infect and complete the life cycle at similar levels as wild-type cells,
despite being extremely sensitive to DNA-damage agents (Kojic et
al., 2002). We favor alternative explanations related to regulation
of the fungal cell cycle inside the plant. U. maydis proliferates inside
the plant as a dikaryotic hyphae. In most dikaryotic Basidiomycetes,
the proper distribution of the two genetically distinct nuclei during
Fig. 10. Virulence of chk1 cells. (A)Quantification of symptoms in maize
plants after infection with wild-type (control) or chk1 mutant crosses.
(B)Images of Chlorazole-Black-E-stained control and chk1 hyphae growing
inside the plant tissue 1 week after infection. Scale bar: 15m. (C)Images of
infected plants. Images show typical large tumors found in wild-type crosses
and the small tumors found in mutant crosses (left). When male flowers were
infected, wild-type crosses produce hypertrofied anthers, whereas limited
deformation is observed in mutant infections (right).
mitotic cell division is ensured by the formation of auxiliary clamp
cells, where one nucleus is entrapped and therefore mitosis occurs
in two distinct cell compartments (Casselton, 2002). In this particular
manner of division, an accurate control of the cell cycle is predicted.
Interestingly, work performed in Coprinopsis cinerea and
Schizophyllum commune indicated a role of homeoproteins from
the b-factor family in this process (Brown and Casselton, 2001). It
is tempting to speculate that Chk1 has a role in this process; its
absence might therefore affect the ability of the dikaryotic cells to
divide properly and therefore proliferation might be affected.
It has become increasingly clear that elements from the DNAdamage response cascade can be used, even in the absence of
apparent DNA damage, to modulate cell cycle progression during
developmental processes such as midblastula transition in
Drosophila embryos (Sibon et al., 1997) or in the asynchronous
division at two-cell-stage C. elegans embryos (Brauchle et al., 2003).
The surprising finding that a protein involved in DNA damage
responses has a role in a fungal developmental process mirrors these
previous results. In addition, our results reinforce the emerging idea
Virulence-induced G2 arrest in smuts
that checkpoint kinases might have roles other than in the DNAdamage response, since they can interact with the cell cycle
machinery. This has been proved by the involvement of Chk2-like
kinases in the pathway linking circadian and cell cycles, in both
mammals and fungi (Gery et al., 2006; Pregueiro et al., 2006).
Materials and Methods
Strains and growth conditions
Ustilago maydis strains are listed in supplementary material Table S1 and are derived
from FB1 and FB2 backgrounds (Banuett and Herskowitz, 1989). Media were
prepared as described (Holliday, 1974). Controlled expression of genes under the
crg1 and nar1 promoters was performed as described previously (Brachmann et al.,
2001; Garcia-Muse et al., 2004). FACS analyses were described previously (GarciaMuse et al., 2003).
Journal of Cell Science
DNA, RNA and protein analysis
U. maydis DNA isolation was performed as previously described (Tsukuda et al.,
1988). RNA isolation and northern analysis were performed as described previously
(Alvarez-Tabares and Perez-Martin, 2008; Castillo-Lluva and Perez-Martin, 2005).
Protein extraction and western blotting were also performed as described previously
(Alvarez-Tabares and Perez-Martin, 2008; Garrido et al., 2004; Pérez-Martín, 2009).
To purify Cdk1 complexes and analyze their kinase activity, previously described
protocols were followed (Garcia-Muse et al., 2004). All quantification was done using
a Phosphorimager (Molecular Dynamics). To detect the phosphorylated and nonphosphorylated forms of Cdk1, commercial antibodies were used, as described
(Sgarlata and Perez-Martin, 2005a). Primary antibody was followed by a secondary
anti-rabbit antibody conjugated to horseradish peroxidase and immunoreactive
proteins were visualized using a chemiluminescent substrate. The chemiluminescent
signal was analyzed using ChemiDoc XCS+ (Molecular Imager, Bio-Rad).
Plasmid and strain constructions
To construct the different strains, transformation of U. maydis protoplasts with the
indicated constructions was performed as described previously (Tsukuda et al., 1988).
All fluorescent protein fusions were already described: GFP-Tub1 (Steinberg et al.,
2001); Cut11-RFP (Perez-Martin, 2009); Cdc25-3GFP (Mielnichuk and PerezMartin, 2008); Chk1-3GFP (Perez-Martin, 2009); GFP-Rad51 (Kojic et al., 2008) (a
gift from William K. Holloman, Cornell University, NY). To express cdk1 alleles
under nar1 promoter control and wee1 under nar1 or scp promoter control, previously
described vectors were used (Sgarlata and Perez-Martin, 2005a). Plasmids to insert
the cdc25AAA allele and the conditional bmh1crg1 allele have been described
(Mielnichuk and Perez-Martin, 2008). Disruption and tagging of chk1 was performed
as described (Perez-Martin, 2009).
To express cdc25 under dik6 promoter control, we exchanged the crg1 promoter
from pRU11-Cdc25 plasmid (Sgarlata and Perez-Martin, 2005b) with the dik6
promoter from the plasmid pCLB1dik6 (Flor-Parra et al., 2006) resulting in the pDik6Cdc25 plasmid. To express NLS-GFP under dik6 promoter control, the otef2 promoter
from plasmid pnGFP (Straube et al., 2001) (a gift from Gero Steinberg, University
of Exeter, Exeter, UK) was exchanged with the dik6 promoter from the plasmid
pCLB1dik6 (Flor-Parra et al., 2006) resulting in the plasmid pDik6-NLSGFP.
Microscopy
Samples were mounted on microscope slides and visualized in a Nikon eclipse 90i
microscope equipped with a Hamamatsu ORCA-ER CCD camera. All the images in
this study are single planes. Standard DAPI, GFP and Rhodamine filter sets were
used for epifluorescence analysis. The software used with the microscope was
MetaMorph 7.1 (Universal Imaging, Downingtown, PA). Images were further
processed with Adobe Photoshop 8.0.
To quantify the ratio of the nuclear intensity (N) to the cytoplasmic intensity (C)
of Cdc25-GFP signal procedures already described were followed (Mielnichuk and
Perez-Martin, 2008). Briefly, the intensity of the nuclear and cytoplasmic signal was
determined by measuring pixel intensity in the nucleus and of an equivalent area in
the cytoplasm, and the ratio was determined. 20 cells were quantified for each
experiment. To quantify Rad51-GFP foci formation, we followed the procedures
described previously (Kojic et al., 2008).
Infection assays
Plant infection and charcoal assays were described previously (Flor-Parra et al., 2007).
Staining of infected plant samples with Chlorazole Black E was done as described
previously (Brachmann et al., 2003).
We thank W. K. Holloman (Cornell University, Ithaca, NY) and G.
Steinberg (University of Exeter, Exeter, UK) for providing plasmids.
Prof. Holloman is also thanked for critical reading and inspiring
discussions. N.M. was supported by JAE. This work was supported by
a Grant from the Spanish Government (BIO2008-04054).
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