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The Induction of the Mating Program in the Phytopathogen
Ustilago maydis Is Controlled by a G1 Cyclin W
Sonia Castillo-Lluva and Jose
´Pe
´rez-Martı
´n
1
Department of Microbial Biotechnology, Centro Nacional de Biotecnologı
´a, Consejo Superior de Investigaciones
Cientı
´ficas, Campus de Cantoblanco, Universidad Auto
´noma de Madrid, 28049 Madrid, Spain
Our understanding of how cell cycle regulation and virulence are coordinated during the induction of fungal pathogenesis is
limited. In the maize smut fungus Ustilago maydis, pathogenesis and sexual development are intricately interconnected.
Furthermore, the first step in the infection process is mating, and this is linked to the cell cycle. In this study, we have
identified a new G1 cyclin gene from U. maydis that we have named cln1. We investigated the roles of Cln1 in growth and
differentiation in U. maydis and found that although not essential for growth, its absence produces dramatic morphological
defects. We provide results that are consistent with Cln1 playing a conserved role in regulating the length of G1 and cell size,
but also additional morphological functions. We also present experiments indicating that the cyclin Cln1 controls sexual
development in U. maydis. Overexpression of cln1 blocks sexual development, while its absence enables the cell to express
sexual determinants in conditions where wild-type cells were unable to initiate this developmental program. We conclude
that Cln1 contributes to negative regulation of the timing of sexual development, and we propose the existence of a negative
crosstalk between mating program and vegetative growth that may help explain why these two developmental options are
incompatible in U. maydis.
INTRODUCTION
Developmental decisions leading to differentiation often require
the cell cycle to be reset and the induction of new morphogenetic
programs. However, understanding how growth and cell cycle
progression are coordinately regulated during development still
remains a challenge. Furthermore, there is little information
available regarding the relationship of these processes with the
induction of the virulence programs in pathogenic fungi. Hence,
the role of cell cycle regulators as true virulence factors has still to
be defined. Nevertheless, in the maize smut fungus Ustilago
maydis, the different morphological changes that the fungal cells
undergo during the pathogenic process indicate that cell cycle
control is closely associated to these transitions (for a review, see
Kahmann and Ka
¨mper, 2004).
The first step in the infection process is mating, and this is linked
to cell cycle. Indeed, when exposed to sexual pheromones,
U. maydis cells undergo arrest in G2 phase, prior to the fusion of
the cytoplasms, and then form the infective dikaryotic filament
(Garcı
´a-Muse et al., 2003). Once this filament enters the plant, the
fungal cells proliferate, forming filaments in which the septa
partition the cell compartments so that each contains a pair of
nuclei (Snetselaar and Mims, 1992; Banuett and Herskowitz,
1996). This morphological transition is clearly connected to cell
cycle control; hence, U. maydis is perfectly suited to analyze the
relationships between cell cycle, morphogenesis, and pathoge-
nicity (Basse and Steinberg, 2004). Two distinct cyclin-dependent
kinase (Cdk)-cyclin complexes are responsible for the different cell
cycle transitions in U. maydis. While Cdk1-Clb1 is required for the
G1/S and the G2/M transitions, the Cdk1-Clb2 complex is specific
for the G2/M transition (Garcı
´a-Muse et al., 2004).
Whether cell cycle regulators may play a role in the pathoge-
nicity of U. maydis has recently been addressed. When the
transcription of mitotic cyclins is manipulated, hyphal proliferation
within the plant is affected and the fungal cells produced
are unable to successfully infect the plant (Garcı
´a-Muse et al.,
2004). Furthermore, we described that mutation of the Fizzy-
related anaphase promoting factor (APC) activator, Cru1, pro-
voked defects at different stages of U. maydis plant infection
(Castillo-Lluva et al., 2004). For instance, in Dcru1 cells, a low level
of expression of the pheromone-encoding gene mfa1 resulted in
deficiency in mating and thereby a low frequency of dikaryotic
infective filament formation. In addition, proliferation of the mutant
fungus inside the plant was also affected, resulting in the inability
to induce tumors in plants (Castillo-Lluva et al., 2004).
Like other Fizzy-related proteins in different organisms, Cru1
controls the length of G1 in U. maydis. Therefore, we proposed
that an accurate control of the length of G1 could be a de-
terminant of virulence in U. maydis. If this is the case, an exciting
challenge would be to establish whether mutations in other
regulators of G1 also affect the pathogenicity of U. maydis cells.
Appealing candidates that should be studied include the genes
encoding G1 cyclins.
In Saccharomyces cerevisiae, the length of G1 depends on the
presence of G1 cyclins that induce the G1/S transition when
1
To whom correspondence should be addressed. E-mail jperez@cnb.
uam.es; fax 34-91585-4506.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Jose
´Pe
´rez-Martı
´n
(jperez@cnb.uam.es).
W
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.105.036319.
The Plant Cell, Vol. 17, 3544–3560, December 2005, www.plantcell.org ª2005 American Society of Plant Biologists
coupled to their Cdk partner (Murray and Hunt, 1993). One such
G1 cyclin, Cln3, is strongly associated with the size threshold for
division and, hence, with G1 length. When the expression of
CLN3 is augmented, the length of G1 is shortened, and cells
divide at a smaller than normal size. By contrast, when CLN3 is
deleted, the G1 phase is delayed, and cells reach a larger than
normal size before dividing (Cross, 1988; Nash et al., 1988).
However, not all G1 cyclins are dedicated to G1 length control.
The other two G1 cyclins from S. cerevisiae, Cln1 and Cln2, play
a major morphogenetic role, inducing bud formation and cell
polarization (Lew and Reed, 1993).
The human pathogen Candida albicans also contains homo-
logues of G1 cyclins, including Cln1/Ccn1, Hgc1, and Cln3 (Loeb
et al., 1999b; Zheng et al., 2004; Bachewich and Whiteway, 2005;
Chapa y Lazo et al., 2005), but it seems that G1 cyclin homo-
logues in this organism have evolved important roles in hyphal
morphogenesis as opposed to cell cycle progression. Deletion of
CLN1 or HGC1 results in defects to maintain hyphal growth (Loeb
et al., 1999b; Zheng et al., 2004), while depletion of Cln3 causes
yeast cells to arrest in G1, increase in size, and then develop into
hyphae and pseudohyphae (Bachewich and Whiteway, 2005;
Chapa y Lazo et al., 2005). Interestingly, repressing CLN3 in
environment-induced hyphae did not inhibit growth or cell cycle,
suggesting that in C. albicans, yeast and hyphal cell cycles may
be regulated differently.
Finally, in other organisms, such as Schizosaccharomyces
pombe, no clear role has been ascribed to the G1 cyclin Puc1,
which is related to Cln3 at the sequence level, but it seems to play
a minimal role in cell cycle, most likely because its activity
is masked by that of the two mitotic cyclins, Cig1 and Cig2
(Martı
´n-Castellanos et al., 2000).
In our attempts to clarify the connection between the cell cycle
and virulence in smut fungi, we have identified a new G1 cyclin
gene from U. maydis that we named cln1. We found that Cln1
is not essential but is required for normal morphogenesis and
cell separation. Repression of cln1 resulted in G1 delay and
cell enlargement, while high levels of cln1 expression induced
hyphal-like growth. These results were consistent with Cln1
playing a conserved role in regulating the length of G1 and cell
size, but also additional morphological functions in influencing
polar growth and cell separation. We also present experiments
suggesting that the cyclin Cln1 controls sexual development in
U. maydis. Our results indicate that U. maydis cells have
established a negative crosstalk between vegetative growth
and mating to make these cell choices incompatible.
RESULTS
Identification of a G1 Cyclin-Related Protein in U. maydis
To determine whether G1 cyclins exist in U. maydis, we used
S. cerevisiae Cln1, Cln2, and Cln3 cyclin box sequences to search
the U. maydis genome database (http://www.broad.mit.edu/
annotation/fungi/ustilago_maydis/index.html). From this ap-
proach, we obtained the sequence of a single gene that we
named cln1. The conceptual translation of the cln1 sequence
produces a putative protein of 520 amino acids (accession
number DQ017758) that groups with members of the G1-type
cyclin family, including putative G1 cyclins from other basidio-
mycetes (Figure 1A). The predicted protein contained one cyclin
box near the N terminus, which shared 73 to 30% sequence
similarity to that of other G1 cyclins characterized in fungi (Figure
1B; see Supplemental Figure 1 online). We also found a PEST
domain at the C terminus of U. maydis Cln1 (Figure 1B). The
PEST domain, found in all three budding yeast G1 cyclins as well
as in S. pombe Puc1 (Tyers et al., 1992; Forsburg and Nurse,
1994), was originally identified as a potential determinant of
protein instability on the basis of its frequent occurrence in un-
stable proteins (Rechsteiner and Rogers, 1996).
Comparison of genomic and cDNA sequences indicated that
cln1 was intronless. Interestingly, the cln1 mRNA 59leader
contains several salient features, such as its unusual length
(551 nucleotides) and the presence of two short upstream open
reading frames (uORFs; Figure 1C). A short uORF in the 59leader
of the S. cerevisiae CLN3 mRNA has been shown to rate-limit
Cln3 production at the translation initiation step by a leaky
scanning mechanism (Polymenis and Schmidt, 1997), suggest-
ing that a similar mechanism could operate in the regulation of
U. maydis cln1 expression.
Cln1 Is a G1-Type Cyclin
We first investigated whether the expression of cln1 fluctuated
through the cell cycle. RNA gel blot analysis was performed on
total RNA isolated from cultures of G1 phase–enriched wild-type
cells or cells arrested in S phase (in the presence of hydroxyurea)
or in M phase (benomyl). We found high levels of cln1 mRNA in
G1-enriched cells (Figure 2A), which dramatically decreased in S
phase–arrested cells. As a control, we followed the expression
levels of clb1, which encodes an essential mitotic cyclin that
reached peak expression in S phase (Garcı
´a-Muse et al., 2004).
This result was consistent with the preferential expression of cln1
during G1. To analyze whether the levels of cln1 mRNA expres-
sion mirror that of the protein, we exchanged the native cln1 gene
by a modified allele that encodes a C-terminal end myc-tagged
Cln1 protein. The resulting strain showed no apparent defects
with respect to a wild-type strain (data not shown), indicating
that the tagged protein was fully functional. However, we were
unable to detect the Cln1 protein directly from crude extract
(data not shown). To circumvent this problem, we used an
immunoprecipitation-based enrichment step, and we observed
the presence of Cln1 protein in immunoprecipitates obtained
from asynchronous and G1-enriched cultures, while only trace
amounts were detected in immunoprecipitates obtained from
S- and M-arrested cells (Figure 2B). From these results, we
believe that the levels of Cln1 protein reproduce that of cln1
mRNA expression; thus, it follows that the Cln1 protein could
exert its main influence in G1 phase.
We also wished to prove that Cln1 might interact with Cdk1,
the catalytic subunit of the mitotic Cdk in U. maydis (Garcı
´a-Muse
et al., 2004). For this, proteins were precipitated from extracts of
wild-type cells and cells expressing high levels of the functional
myc-tagged version of Cln1. Initially, the protein extracts were
incubated with agarose beads coupled to the S. pombe protein
Suc1, which is known to bind specifically to Cdk1 with high
G1 Cyclin Controls Mating in Smuts 3545
Figure 1. Cln1 Is a G1-Type Cyclin.
3546 The Plant Cell
affinity (Ducommun and Beach, 1990; Garcı
´a-Muse et al., 2004).
The precipitates were then separated by SDS-PAGE and immu-
noblotted with anti-PSTAIRE (which detects Cdk1) and anti-MYC
antibodies. Both Cdk1 and Cln1-myc were recovered in the
fraction of proteins bound to Suc1-beads (Figure 2C, left panel).
The presence of Cdk1 was also analyzed in protein extracts
precipitated with anti-MYC antibodies (Figure 2C, right panel).
While these antibodies coprecipitated Cdk1 with Cln1-myc,
neither Cln1 nor Cdk1 was detected in precipitates from wild-
type cell extracts. In addition, the precipitate from tagged strains
exhibited histone H1 kinase activity (data not shown). These
results demonstrate a physical association between the Cln1
protein and Cdk1, further evidence that Cln1 can be considered
as a definitive cyclin.
Finally, we also investigated whether U. maydis cln1 could
complement the absence of G1 cyclins in S. cerevisiae,as
described for other G1-related cyclins (Forsburg and Nurse,
1991; Whiteway et al., 1992; Sherlock et al., 1994; Zheng et al.,
2004). We tested this in an S. cerevisiae strain in which the CLN1
and CLN2 genes had been deleted and where the CLN3 gene
was under the control of GAL1 promoter. The viability of this
strain is conditional and galactose dependent (Tyers et al., 1993).
We introduced into this strain the U. maydis cln1 gene under the
control of the constitutive GPD1 promoter on a centromeric
plasmid. As positive control, we used a centromeric plasmid
carrying the S. cerevisiae CLN3 locus. All the strains grew on
solid medium carrying galactose, a condition that allows GAL1-
CLN3 expression, while when galactose was replaced by glu-
cose, only the strains carrying the CLN3 or cln1 plasmids grew,
although the strain expressing cln1 grew considerably more
slowly (Figure 2D). The same test in liquid medium produce
similar results. Interestingly, ;17% of the cells carrying the
vector expressing the U. maydis cln1 gene exhibited an elon-
gated morphology (Figure 2E). The polarized morphology of the
S. cerevisiae cells expressing U. maydis cln1 might be a reflection
of some intrinsic ability of this protein in promoting morphogen-
esis (see below). Although these results show that U. maydis cln1
may be only partially functional in S. cerevisiae, collectively,
these data support the notion that the cln1 gene encodes a G1
cyclin in U. maydis.
Cln1 Is Not Essential but Is Required for Normal
Morphogenesis and Cell Separation
To analyze the function of Cln1, we inactivated one cln1 allele in
the diploid FBD11 strain, replacing it with a carboxin resistance
cassette (producing the Dcln1 null allele). When the meiotic
offspring was analyzed after sporulation, half the resulting
population was resistant to carboxin and carried the null allele,
indicating that cln1 is not essential for growth. However, in
contrast with the characteristic cigar-shaped wild-type morphol-
ogy, a cln1-deficient strain generated very thick and irregularly
shaped cell aggregates (Figure 3A). In these cell aggregates,
most cells did not separate, and they form septa that could be
stained with calcofluor (Figure 3B). Older cells often lost their
polarity and became rounded, although the cells at the periphery
of the aggregates remained elongated. We also found that some
cells accumulated several nuclei (Figure 3A, arrowheads).
Repression of cln1 Results in G1 Delay
In S. cerevisiae, the absence of Cln3 causes a delay in G1 and an
increase in cell size (Cross, 1988; Nash et al., 1988). The deletion
of cln1 in U. maydis resulted in enlarged cells. However, because
of the cell separation defect producing cell aggregates, we were
unable to correlate the absence of Cln1 with a delay in G1 (that
could be measured as an increase in 1C DNA content in the cell
population by fluorescence-activated cell sorter[FACS] analysis).
To circumvent this difficulty, we constructed a conditional allele,
cln1
nar
, in which the cln1 was placed under the control of the nar1
promoter (Brachmann et al., 2001). This enabled the down-
regulation of cln1 expression to be controlled by switching from
nitrate- to ammonium-containing medium (Figure 4A). To avoid
any putative negative regulation exerted by the long 59untrans-
lated region, in this chimeric construction, we removed the native
cln1 mRNA leader region, leading to a shorter transcript when
compared with the wild-type transcript. No defects in cell growth
were apparent in the resulting strain under permissive conditions
(data not shown). However, when shifted to restrictive condi-
tions, cells start to enlarge (cf. with wild-type cells, Figure 4B,
inset) and to have cell separation defects (Figure 4B, 3 h). After an
extended period of time, cell aggregates started to form in which
the older cells became rounded, while the new ones remained
elongated (Figure 4B, 6 h). Prolonged incubations (>24 h) lead to
the appearance of large aggregates that were reminiscent of
those seen in Dcln1 cells (data not shown).
To address whether cln1-deficient cells have an elongated G1
phase, we grew wild-type and cln1
nar
cells in permissive con-
ditions (nitrate-containing minimal medium) until early exponen-
tial phase, and then cells were arrested at M phase with benomyl
and transferred to repressive conditions for cln1 expression
(complete medium [CMD]) for 1 h. After being released in
Figure 1. (continued).
(A) A dendrogram of G1 cyclins created by the distance-based minimum evolution method based on 500 replicates. Branching distances are given, and
the scale bar denotes substitutions per site. Sc, Saccharomyces cerevisiae;Ca,Candida albicans; Sp, Schizosaccharomyces pombe; Cc, Coprinus
cinerea;Cn,Cryptococcus neoformans; Um, Ustilago maydis.
(B) Scheme of the Cln1 protein in relation to the described G1 cyclins from other fungi. The cyclin boxes are shown in black, and the percentage values
inside each box represent the sequence identity to U. maydis Cln1 cyclin box. The putative PEST domains are indicated as white boxes. aa, amino
acids.
(C) Schematic representation of cln1 gene structure, showing the cln1 ORF and the uORFs. Relative positions of nucleotide sites including the 59end of
the mRNA and of the predicted uORF are indicated and numbered with respect to the initiation codon in cln1 coding sequences.
G1 Cyclin Controls Mating in Smuts 3547
benomyl-free CMD medium, the DNA content was followed as
these cells proceeded through mitosis and entered in G1 and S
phases (Figure 4C). We observed that upon release, mutant cells
were delayed in G1 phase with respect to wild-type cells (cf. the
1C population on wild-type and cln1
nar
cells at 120 min). These
results were consistent with a role of Cln1 promoting the tran-
sition through G1 phase.
High Levels of cln1 Expression Induce Hyphal-Like Growth
G1 cyclins are thought to be involved during G1 in coordinating
cell growth and division. For instance, an increase in the amount
of CLN3 in S. cerevisiae results in cells that divide when they are
smaller than normal (Cross, 1988; Nash et al., 1988). We in-
troduced an extra copy of cln1 into U. maydis cells under the
Figure 2. Characterization of the Cln1 Protein.
(A) Levels of cln1 expression at different stages of the cell cycle. RNA extracted from wild-type FB1 cells arrested at S phase, arrested at M phase,
enriched in G1 phase, or growing asynchronously (As) was analyzed by RNA gel blotting. The filters were hybridized with probes for cln1,clb1, and 18s
rRNA as a loading control.
(B) Cln1 protein levels at different stages of the cell cycle. Protein extracts from SONU59 (a1 b1 cln1-myc) cultures arrested at S phase, arrested at M
phase, enriched in G1 phase, or growing asynchronously (As) were immunoprecipitated with anti-MYC antibodies, and the precipitates were separated
by SDS-PAGE and immunoblotted with anti-MYC to detect Cln1-myc.
(C) Cln1 associates with Cdk1. Lysates prepared from wild-type FB1 cells (cln1) and SONU58 cells expressing a myc-tagged version of Cln1 (cln1-myc)
were incubated with Suc1 beads to pull down Cdk1 (left panel), or the lysates were immunoprecipitated with anti-MYC antibodies to pull down Cln1-
myc (right panel). The whole-cell lysates, as well as the precipitates, were separated by SDS-PAGE and immunoblotted with anti-PSTAIRE and anti-
MYC to detect Cdk1 and Cln1-myc, respectively.
(D) U. maydis cln1 supports the growth of an S. cerevisiae strain without endogenous G1 cyclins. The yeast strain BF305-15#21 (cln1D,cln2D,and
GAL1-CLN3) was transformed with a control plasmid (vector), a plasmid expressing S. cerevisiae CLN3, or a plasmid expressing U. maydis cln1.The
resulting strains were spotted in solid medium containing 2% galactose or glucose as a carbon source, and they were incubated at 308Cfor2d.
(E) Liquid cultures of the above strains were grown in minimal medium with 2% galactose to midexponentia l phase and then were transferred to minimal
medium with 2% glucose and incubated for 6 h. Note that some of the cln1-expressing cells exhibited an elongated morphology. DAPI, 49,6-diamidino-
2-phenylindole.
3548 The Plant Cell
control of the crg1 promoter, which can be induced by arabinose
(Bottin et al., 1996). In this construction, we removed the long 59
mRNA leader region, producing a shorter transcript, and the
resulting protein was myc-tagged at its C-terminal end. In cells
carrying this construct, mRNA transcribed from the crg1 pro-
moter (Figure 5A, crg:cln1) was detected after <1 h of induction,
and the accumulation of this RNA reached a peak of expression
;25 times the levels of native cln1 mRNA expression. The
increase in mRNA expression was followed by protein accumu-
lation (Figure 5B). Strikingly, these high levels of cln1 expression
from the crg1 promoter resulted in the downregulation of the
mRNA transcribed from the native promoter (Figure 5A, cln1).
This is suggestive of the existence of autoregulatory feedback
inhibition as reported for other cyclins (Ayte et al., 2001).
Unexpectedly, cells overexpressing cln1 displayed hyperpo-
larized growth after the shift to inducing conditions (Figure 5C).
The cells started to grew apically, and after 3 h, they clearly
evaginated outgrowths carrying a single nucleus each, with
a 2C DNA content (Figure 5D). Subsequently, the filaments be-
gan to undergo mitosis, and by 8 h, approximately half of the cell
population contained four nuclei. Cells not overexpressing cln1
and growing in arabinose-containing medium would have gone
through approximately four cell divisions. Thus, cln1-induced
filaments appear to be elongated buds that can progress through
the nuclear cell cycle, albeit with an apparent delay. Prolonged
incubation (24 h) resulted in the development of highly elongated
cells that resembled hyphae, with several nuclei evenly distri-
buted along the entire length of the filament. Staining of the cells
with calcofluor showed the presence of septal cross-walls
between hyphal compartments (Figure 5E).
Cln1 Is a Nuclear Protein That Also Localizes to
the Hyphal Tip
The previous data suggesting a morphological role of Cln1
prompted us to analyze the subcellular localization of Cln1. For
this, we constructed a strain that contained green fluorescent
protein (GFP) fused to the C terminus of the endogenous cln1
gene. This strain had a wild-type appearance, although we were
unable to detect Cln1-GFP by epifluorescence. Therefore, for
in vivo localization, weexpressed this protein fusion under control
of the crg1 promoter and analyzed the cells during the first 2 h.
The Cln1-GFP fusion protein localized to the nucleus (Figure 6A),
as it was expected for a cyclin protein. Strikingly, we also found
fluorescent single spots that were associated to the region just
behind the hyphal tip (Figure 6B). These spots were motile,
showing random short-distance mobility (S. Castillo-Lluva, un-
published data). Currently, we do not have a satisfactory ex-
planation for this localization, but it was compatible with a
morphogenetic role of Cln1 in U. maydis.
High Level of Cln1 Represses the Induction of
the mfa1 Gene
The above results were consistent with Cln1 having a conserved
role in regulating the length of G1 and cell size, but also additional
morphological functions. Because our previous suggestion that
induction of the sexual program (as measured by the expression
of the mfa1 gene, encoding the pheromone precursor) was
related to G1 length (Castillo-Lluva et al., 2004), we wanted to
examine the relationships between Cln1 levels and mfa1 ex-
pression. Since the culture medium has a strong influence in the
mfa1 expression (it is expressed in complete- and minimal-
based media but not in yeast extract peptone (YEP)-based
medium; Spellig et al., 1994), we constructed a new cln1
conditional allele, cln1
crg
, in which the endogenous cln1 gene
was placed under the control of the crg1 promoter. This enabled
us to downregulate or to overexpress the cln1 gene in any
medium by switching the carbon source from glucose to arab-
inose, respectively (Figure 7A). In glucose-containing media,
these cells displayed the altered morphology observed in a cln1-
deficient strain, while in arabinose-containing media, they had
the hyphal-like growth observed when cln1 was overexpressed
ectopically (data not shown). We found that depletion of Cln1
enables the cells to express mfa1 even on media like YEP-based
Figure 3. Cln1 Is Required for Normal Morphogenesis and Cell Sepa-
ration.
(A) Morphology of Dcln1 cells. Wild-type (FB1) and Dcln1 (SONU76) cells
grown in CMD medium until the exponential phase was observed by
phase contrast and by epifluoresence to visualize the DAPI staining.
Arrowheads indicate cells carrying more than one nucleus. Bars ¼
10 mm.
(B) Chitin distribution in Dcln1 cells. Calcofluor (CFW) detects chitin at
cell tips in wild-type FB1 cells, while in SONU76 cells, it stains the septa.
Bars ¼10 mm.
G1 Cyclin Controls Mating in Smuts 3549
medium, for which respective wild-type strain was unable to
express this gene (Spellig et al., 1994; Castillo-Lluva et al., 2004).
By contrast, overexpression of cln1 abrogates the mfa1 expres-
sion in media like CMD (Figure 7A), where mfa1 is efficiently
expressed in wild-type strains (Spellig et al., 1994). To study in
detail this correlation, we grew cln1
crg
cells in CMD, conditions in
which no expression of cln1 was observed and a strong signal
in mfa1 was apparent (Figure 7B), and transferred to arabinose-
containing CMD (CMA). Consistently, we found strict inverse
correlation between the cln1 and mfa1 mRNA levels (Figure 7B).
Transfer of wild-type cells from CMD to CMA has no influence on
the levels of mfa1 expression (data not shown).
The negative role of Cln1 in the expression of mfa1 contrasts
with the positive role that the Fizzy-related protein Cru1 has in
pheromone gene expression (Castillo-Lluva et al., 2004). Since
Cru1 is also implicated in G1 regulation, we decided to examine
the relationships between Cln1 and Cru1 with respect to mfa1
expression. Therefore, we deleted cln1 in cells already lacking
the cru1 gene. While cells lacking cln1 showed higher levels of
mRNA than wild-type cells, Dcru1 cells were unable to express
mfa1. Strikingly, cells lacking both regulators were unable to
express mfa1 (Figure 7C), indicating a dominant effect of Dcru1
mutation over of Dcln1.
cln1 Expression Is Regulated by the cAMP/Protein Kinase A
and Mitogen-Activated Protein Kinase Pathways
The cAMP/protein kinase A (PKA) and mitogen-activated protein
kinase (MAPK) pathways regulate the expression of the phero-
mone gene (Kaffarnik et al., 2003). The relationship between Cln1
levels and pheromone gene expression led us to assess whether
these pathways also control cln1 expression. For this, we
Figure 4. Cln1 Regulates G1 Length.
(A) Levels of cln1 mRNA in the cln1
nar
conditional strain. Cultures of wild-type FB1 and conditional strain SONU64 (cln1
nar
) were grown under permissive
conditions (minimal medium with nitrate as nitrogen source [MM-NO
3
]) to an OD
600
of 0.2 and shifted to permissive (MM-NO
3
) or restrictive (CMD)
conditions for 4 h. The RNA was then extracted and analyzed by RNA gel blotting using a probe for cln1 and for 18s rRNA as a loading control.
(B) Morphology of conditional cln1 mutant cells at different times after transfer to restrictive conditions (CMD). Inset shows a wild-type cell at the same
magnification. Bars ¼10 mm.
(C) Analysis of G1 length in the absence of Cln1. Wild-type and cln1
nar
cells were grown under permissive conditions (MM-NO
3
)toanOD
600
of 0.2 and
then shifted to CMD medium amended with benomyl for 90 min. Cells arrested at G2/M transition were released in benomyl-free CMD medium, and
samples were taken at the indicated times. FACS analysis was performed with these samples.
3550 The Plant Cell
examined its mRNA levels in Dadr1 and Dfuz7 mutants, in which
the cAMP/PKA and the pheromone MAPK pathways are blocked,
respectively (Banuett and Herskowitz, 1994; Du
¨rrenberger et al.,
1998; Andrews et al., 2000). We also analyzed the cln1 expres-
sion in cells carrying the Dubc1 mutation, in which the PKA
pathway is constitutively activated (Gold et al., 1994), and in cells
expressing an activated allele of the Fuz7 mitogen-activated
protein kinase kinase, fuz7
DD
(Mu
¨ller et al., 2003), under a regu-
latable promoter.
When the regulatory subunit of PKA, ubc1, was deleted, the
resulting constitutive activation of the cAMP/PKA pathway
downregulated the cln1 expression (Figure 8A). Conversely,
a deletion of the PKA catalytic subunit that interrupts the pathway
produced an increase in the cln1 mRNA levels (Figure 8A). With
respect to the pheromone MAPK pathway, we found that de-
letion of the fuz7 gene increases cln1 expression approximately
threefold (Figure 8A), while the expression of the constitutive
active fuz7
DD
allele strongly repressed the cln1 expression
Figure 5. Ectopic Expression of cln1 Induces Polar Growth.
(A) RNA gel blot analysis of wild-type FB1 cells and SONU58 cells expressing an ectopic copy of cln1-myc under the control of the crg1 promoter. Cells
were grown under repressing conditions (CMD with glucose) to an OD
600
of 0.2 and then shifted to inducing conditions (CMD with arabinose). Samples
were taken at the times indicated. Note the different migration of the mRNA species generated from the native cln1 promoter (cln1 arrowhead) and the
crg1-cln1 fusion (crg:cln1 arrowhead) as a result of the different transcriptional start point.
(B) Cln1 protein levels in SONU59 cells growing under inducing conditions (CMD with arabinose). Protein extracts obtained at the time indicated after
shift to inducing conditions were separated by SDS-PAGE and immunoblotted with anti-MYC and anti-PSTAIRE to detect Cln1-myc and Cdk1 as
loading controls. Cdk2 is the homolog of S. cerevisiae Pho85 that is also recognized with anti-PSTAIRE.
(C) Time course of the morphological changes in SONU58 cells grown in inducing conditions. Note that after 24 h, the cells form filaments carrying
several nuclei. Bars ¼20 mm.
(D) Flow cytometry analysis of SONU58 cells (Pcrg:cln1) expressing high levels of cln1 using wild-type FB1 cells as controls. Samples were taken for
FACS analysis at the times indicated after transfer to induction conditions (CMD with arabinose).
(E) Calcofluor staining of SONU58 cells after 24 h of growth in inducing conditions. At bottom is a Photoshop inverted image to show more clearly the
septum positions (asterisks). Bar ¼40 mm.
G1 Cyclin Controls Mating in Smuts 3551
(Figure 8B). In wild-type cells, the expression of cln1 was not
affected by the transfer from glucose- to arabinose-containing
media (data not shown).
Cln1 and Cru1 Control the Expression of prf1, Encoding
the Master Regulator of Sexual Development
The transcription factor Prf1 receives the information from both
cAMP/PKA and MAPK pathways and activates the expression of
genes involved in sexual development, including the mfa1 gene
(Hartmann et al., 1996; Kaffarnik et al., 2003). Since Cln1 affects
mfa1 expression and it is regulated by the same pathways as
Prf1, we decided to analyze putative relationships between prf1
and cln1.
First, we wondered whether Prf1 negatively regulates cln1
expression. We expressed the constitutive active fuz7
DD
allele in
Dprf1 cells, and we found that the absence of Prf1 did not
suppress the negative regulation over cln1 of an activated MAPK
pathway (Figure 8B).
We also analyzed the level of expression of the prf1 gene
in cells carrying the conditional cln1
crg
allele in induction (CMA)
and noninduction (CMD) conditions as well as in Dcru1 cells.
We observed (Figure 9A) that prf1 mRNA was not detectable in
the absence of cru1 or in conditions of high cln1 expression,
mirroring the mfa1 levels previously observed (Figures 7A and
7C). This result suggests that the absence of prf1 transcription in
these genetic backgrounds might be responsible for the absence
of mfa1 expression in these conditions. To support this interpre-
tation, we put the prf1 gene in these strains under the control of
the regulatable nar1 promoter. We observed mfa1 expression in
Figure 6. Subcellular Location of Cln1-GFP.
(A) SONU84 cells expressing a Cln1-GFP fusion were grown for 2 h in
inducing conditions (CMD with arabinose [CMA]). Cln1-GFP localizes in
the nucleus. Bar ¼20 mm.
(B) Location of Cln1-GFP dots around the hyphal tip. Single dots are
marked with arrows. Because of the different fluorescence intensity
threshold between the nucleus and the rest of the cell, only the tip is
shown. Bar ¼10 mm.
Figure 7. Cln1 Represses Sexual Development.
(A) Effects of the cln1
crg
allele on the expression of the pheromone gene
mfa1 are analyzed by RNA gel blotting. SONU73 cells were grown on
YEP-based and complete media amended with glucose (YPD and CMD)
or arabinose (YPA and CMA) as carbon source until midexponential
phase. The filter was hybridized in succession with probes for cln1,mfa1,
and 18s rRNA, as indicated at left.
(B) Time course induction of cln1 and its effect on mfa1 expression.
SONU73 cells growing in glucose-containing CMD were washed three
times and transferred to arabinose-containing CMD, and samples were
obtained at the indicated times (in hours). RNA was isolated and
submitted to RNA gel blot analysis as above.
(C) Relationships between cru1 and cln1 in pheromone gene expression.
Wild-type cells (FB1), the single Dcru1 (UMP7) and Dcln1 (SONU76)
mutants, and the double Dcru1 Dcln1 mutant (SONU79) were grown in
CMD to midexponential phase. The RNA was then extracted and
analyzed by RNA gel blotting using a probe for mfa1,cru1,cln1,and
for 18s rRNA as a loading control.
3552 The Plant Cell
Dcru1 cells when transcription of the prf1 gene was induced
(Figure 9B). We also were able to bypass the cln1-mediated
repression of mfa1 expression by induced expression of prf1
under Pnar1 control (Figure 9C). These results support the notion
that Cru1 and Cln1 control the induction of sexual development
in U. maydis via the master regulator Prf1.
Absence of Cln1 Affects Cell–Cell Fusion during Mating
Given the connections between cell cycle control and patho-
genesis in U. maydis, we were interested in determining whether
cells deficient in cln1 were able to infect plants. For this, we
infected maize (Zea mays) plants with a mixture of compatible
(i.e., with different aand bloci) Dcln1 strains. While in control
experiments with mixtures of wild-type compatible strains, 95%
of infected plants showed tumor formation, and compatible
Dcln1 strains were completely impaired in pathogenic develop-
ment. Not one of the 74 plants inoculated with the compatible
mutant cells showed tumors (Table 1).
Such a severe defect in virulence led us to investigate the
ability of Dcln1 cells to mate, a prerequisite to initiate the
pathogenic development. The mating reaction in U. maydis can
be easily scored by cospotting compatible strains on solid media
containing charcoal. In these plates, cell fusion and development
of the infective dikaryotic filament resulted in the formation of
a white layer of aerial hyphae on the surface of the growing
colony. While control mating reactions between compatible wild-
type strains produce a clear formation of aerial filaments, when
the strain carrying the Dcln1 allele was cospotted with a wild-
type compatible strain, the formation of the aerial filaments was
attenuated, and additional deficiency was observed when two
compatible Dcln1 strains were mixed together (Figure 10A).
Furthermore, in spite of the fuzzy aspect of small areas in the
mutant mixture, we were unable to detect any dikaryotic filament
in these mixtures (data not shown). The formation of dikaryotic
hyphae requires the fusion of conjugation tubes; therefore, it is
possible that the inability to produce a successful mating in Dcln1
cells is a consequence of defects in cell–cell fusion. To circum-
vent the need for cell fusion, we deleted the cln1 gene in the
solopathogenic strain SG200, which carries the genetic infor-
mation required to form hyphae without a mating partner (Bo
¨lker
et al., 1995). Strikingly, a solopathogenic Dcln1 strain was able to
produce infective hyphae as well as the SG200 strain (Figure
10A). We also observed formation of normal appressoria in the
plant surface (see Supplemental Figure 2 online). Finally, in
Figure 8. The mRNA Levels of cln1 Are Regulated by cAMP/PKA and
MAPK Pathways.
(A) Levels of cln1 mRNA in different signaling mutants. Total RNA from
the strains indicated at the top (WT, FB1; Dadr1, SONU24; Dubc1,
UME18; Dfuz7, FB1Dfuz7) growing in CMD until midexponential phase
was isolated and subjected to RNA gel blot analysis with the probes
indicated at the left.
(B) cln1 is repressed by the MAPK cascade. SONU8 cells carrying an
ectopic copy of fuz7
DD
, a constitutive allele of the Fuz7 MAPK kinase
under the control of Pcrg1 promoter, were grown to an OD
600
of 0.2 in
noninducing conditions (CMD) and then shifted to noninducing condi-
tions (CMD) and inducing conditions (CMA) for 6 h. A derivative strain
(SONU34) without the prf1 gene was used to assess the putative
implication of this transcriptional regulator in the downregulation of
cln1 expression.
Figure 9. Induced Expression of prf1 Bypasses the Cyclin-Mediated
Repression.
(A) Expression of prf1 is under Cln1 and Cru1 control. Wild-type,
SONU73 (cln1
crg
), and UMP7 (Dcru1) cells were grown until midexpo-
nential phase in the indicated media and submitted to RNA gel blot
analysis with a prf1 probe and 18s rRNA as a loading control.
(B) Induced expression of prf1 bypasses the Cru1 requirement for mfa1
expression. Cells carrying a prf1
nar
conditional allele, with (SONU27) or
without a functional cru1 gene (SONU38), were grown in inducing
(MMNO
3
) and noninducing conditions (MMNH
4
)forprf1
nar
expression
until midexponential phase. The RNA was then extracted and analyzed
by RNA gel blotting using a probe for mfa1 and for 18s rRNA as a loading
control.
(C) Effects of the prf1
nar
allele on the expression of the pheromone gene
mfa1 in strains overexpressing cln1. Strains SONU27 (prf1
nar
)and
SONU115 (prf1
nar
cln1
crg
) were grown in the conditions indicated until
midexponential phase, and isolated RNA was submitted to RNA gel
blotting to detect mfa1 transcripts.
G1 Cyclin Controls Mating in Smuts 3553
virulence studies the mutant Dcln1 strain was fully pathogenic
(Table 1).
Since these results indicated that the defects associated with
the absence of Cln1 were prior to cell–cell fusion, we analyzed
the ability of mutant cells to secrete pheromone. For this, we took
advantage of the strain FBD12-17, a tester strain used to check
for pheromone production. If this strain is cospotted together
with cells secreting a1 pheromone, a strong formation of hyphal
growth is induced (Spellig et al., 1994). We spotted, together with
a constant amount of tester cells, increasing amounts of wild-
type and Dcln1 cells, and we found that although mutants cells
were able to secrete pheromone, they were clearly impaired in
this process in comparison with wild-type cells (Figure 10B).
We also analyzed the ability of Dcln1 cells to produce con-
jugative tubes in response to the presence of compatible
pheromone. We treated wild-type and Dcln1 cells with synthetic
a2-mating pheromone, and we found that mutant cells formed
significantly less tubes (Figure 10C) that in general were thicker
Figure 10. Cells Lacking Cln1 Are Impaired in Cell–Cell Fusion.
(A) Effects of absence of cln1 in formation of infective filaments. Mixtures of compatible wild-type (FB1 and FB2), wild-type and cln1 mutants (SONU76
and FB2), and mutant (SONU76 and SONU75) strains (top row) or the solopathogenic strain SG200 and its Dcln1 derivate (SONU130) were spotted in
CMD-charcoal plates. The appearance of white infective filaments was assayed after 24-h incubation at room temperature.
(B) Assay for a1 pheromone production in mutant cells. The 10
4
cells from tester strain FBD12-17 were cospotted with the indicated amount of cells of
wild-type (FB1) and Dcln1 mutant (SONU 76) strains in CMD-charcoal plates. Formation of infective filaments was scored after 24 h at room temperature.
(C) Formation of conjugative tubes in wild-type (FB1) and Dcln1 mutant (SONU 76) cells after incubation in CMD amended with 2.5 ng/mL of synthetic a2
pheromone. A total of 100 cells were counted in each assay.
(D) Morphology of conjugative tubes in FB1 wild-type and Dcln1 mutant (SONU 76) cells. Cells were incubated as above during 8 h. Note that Dcln1
mutant cells are actually composed for more than one cell compartment, explaining the presence of more than one tube per cell.
Table 1. Pathogenicity Assays
Anthocyanin Formation Tumor Formation
Inoculum Genotype Total Percentage Total Percentage
FB1 3FB2 a1 b1 3a2 b2 57/60 95 57/60 95
SONU76 3SONU75 a1 b1Dcln1 3a2 b2 Dcln1 0/74 0 0/74 0
SG200 a1mfa2 bW2bE1 46/52 88 43/52 82
SONU130 a1mfa2 bW2bE1 Dcln1 72/77 93 70/77 90
3554 The Plant Cell
and more irregular than those produced by wild-type cells (Figure
10D), indicating that deletion of cln1 affects induction and
morphology of conjugation tubes.
In summary, haploid Dcln1 cells were nonvirulent mainly
because of a defect in cell–cell fusion that we believe arises at
least from impaired pheromone secretion and conjugative tube
formation.
DISCUSSION
Fungal cells take different developmental options in response to
external signals, and frequently these options are incompatible.
In the phytopathogenic fungus U. maydis, pathogenicity and
sexual development are intimately intertwined, and both are part
of the same developmental program. Entering the mating pro-
gram involves cell cycle arrest, suggesting that to have an active
mitotic cycle could be incompatible with the induction of the
sexual program. This work concerns the relationships between
key molecules involved either in the regulation of G1 phase or in
the activation of the sexual development in U. maydis and
provides some clues as to how smut fungus make these choices
incompatible.
Role of Cln1 in G1 Progression and Morphogenesis
U. maydis cln1 encodes a protein with high sequence similarities
to G1 cyclins from other fungi. This protein can interact with the
catalytic subunit of the mitotic Cdk, and it is able to complement
the lack of G1 cyclins when expressed in S. cerevisiae. We also
showed that the cln1 gene is preferentially expressed in G1
phase. In agreement with this view, cell cycle arrest/release
experiments indicated that cells lacking Cln1 had a delayed G1
phase with respect to wild-type cells. G1 cyclins in fungi and
D-type cyclins in mammalian are thought to be essential regu-
lators that mediate G1/S transition. In S. cerevisiae, there are
three major G1 cyclins: Cln1, Cln2, and Cln3. Although they play
different roles, G1 cyclins in budding yeast are functionally
redundant, since viability is not compromised by the deletion
of any two of the three; however, deletion of all three is lethal
(Richardson et al., 1989). In U. maydis, Cln1 is dispensable to
growth. However, we found no additional sequences in the
available genome sequence with capacity to encode another
G1-like cyclin. It could be possible that additional elements, like
Bck2 in S. cerevisiae, share function with Cln1 in activating G1/S
transition (Epstein and Cross, 1994; Di Como et al., 1995) or that
its activity is masked by other Cdk-cyclin complexes, as it
happens in S. pombe, where the solely G1 cyclin Puc1 is masked
by the activity of the two mitotic cyclins Cig1 and Cig2 (Martı
´n-
Castellanos et al., 2000). In any case, our data indicated that Cln1
plays a conserved role in regulating the length of G1.
Our results also indicated that Cln1 plays additional roles in
morphogenesis. The cell aggregates produced by the absence
of Cln1 are composed of cells that lost their ability to divide by
budding, remain attached after cytokinesis, and often lost their
polarity. By contrast, high levels of Cln1 provoke a strong polar
growth that results in filaments composed of cells separated by
septa. The involvement of G1 cyclins in fungal morphogenesis is
well known. In S. cerevisiae, the G1 cyclins Cln1 and Cln2 play
a role in the establishment of growth polarization and budding
(Benton et al., 1993; Cvrckova and Nasmyth, 1993; Lew and
Reed, 1993), and mutants that enhance or prolong the activities
of Cln1,2-Cdc28 complexes sometimes cause significant bud
elongation (Lew and Reed, 1993; Loeb et al., 1999a). The
different G1-like cyclins from C. albicans plays distinct morpho-
genetic roles. Deletion of CLN1 results in the inability to maintain
hyphal growth under certain conditions (Loeb et al., 1999b), while
deletion of HCG1 prevents hyphal growth under all hypha-
inducing conditions (Zheng et al., 2004). By contrast, deletion
Figure 11. Model of the Negative Relationships between the Cell Cycle
and the Virulence Program in U. maydis.
An active cell cycle represses the induction of the mating program, while
the induction of the mating program arrests the cell cycle. Cell cycle
transmits the negative information to the sexual program most likely via
Cdk1-cyclin complexes that negatively regulate the transcription of prf1,
the master sexual regulator. Environmental information is transmitted via
cAMP/PKA and MAPK pathways to the different elements. Dashed
lines indicate a putative interaction that remains to be identified (see
Discussion).
G1 Cyclin Controls Mating in Smuts 3555
of CLN3 produces hyphal growth in the absence of environmen-
tal inducing conditions (Bachewich and Whiteway, 2005; Chapa
y Lazo et al., 2005). From our data, we infer that in U. maydis, Cln1
could be related to cell polarity and budding, recapitulating some
of the roles of the three different G1 cyclins from S. cerevisiae.
The mechanism by which Cln1 may affect morphogenesis in
U. maydis is unknown. In addition to a nuclear distribution, we
were able to locate Cln1-GFP, when overexpressed, as small
unstable dots in the tip of the filament (Figure 6B). However, in
spite of the dramatic morphological effect observed when over-
expressed, we believe that Cln1 is not directly involved in hyphal-
like promotion in U. maydis. We found that conditions producing
hyphal-like growth in U. maydis, such as the induced expression
of an activated form of the Fuz7 MAPK kinase or the expression
of an active bE/bW heterodimer, were not affected by the
absence of Cln1 (see Supplemental Figure 3 online). Moreover,
we believe that the hyphal-like growth resulting from a continuous
supply of Cln1 through the cell cycle could be the consequence
of the interference with a correct morphogenesis, and this could
be producing a cell cycle delay that leads to hyperfilamentation.
Mitosis or Mating: A Role for G1 Cyclins
In U. maydis, entering the sexual program seems to be incom-
patible with an active mitotic cell cycle. The response to
pheromone and the activation of the mating program induces
a cell cycle arrest in U. maydis (Garcı
´a-Muse et al., 2003),
whereas Cdk1-cyclin complexes seem to be repressive for the
induction of sexual determinants (Figure 11). High levels of cln1
expression impair the expression of mfa1, encoding the pre-
cursor of sexual pheromone a1, while downregulation of cln1
enables the expression of mfa1 even in nutritional conditions that
repress the expression of the amating types. Similarly, we
previously reported that the requirement of the APC activator
Cru1 for mfa1 expression could be bypassed by downregulation
of clb1 expression (Castillo-Lluva et al., 2004), suggesting that
the requirement of Cru1 for pheromone expression is just to
remove Clb1. Several results support that Cru1 and Cln1 are
acting in the same pathway, or at least in the same target. We
showed that the deletion of cln1 failed to rescue the mfa1
expression in Dcru1 cells, suggesting that either the inhibitory
function of Cln1 is upstream of Cru1 activating function or that
the Cru1 functions in parallel with the Cln1 cyclin in regulating
pheromone expression, but Cru1 requirement has a stronger
effect on the process. The inability to express mfa1 seems to be
related to the absence of prf1 expression when Cru1 is absent or
cln1 is overexpressed. Moreover, the effect on mfa1 expression
of the absence of cru1 and the overexpression of cln1 are
suppressed by heterologous expression of prf1. These results
suggest that the expression of prf1 seems to be the target of the
negative cell cycle regulation. The prf1 promoter has previously
been shown to underlie a complex regulation, with at least three
different positive regulators acting on distinct regulatory regions:
Prf1 itself, which binds pheromone response elements
(Hartmann et al., 1996), Crk1, a kinase that acts through the
upstream activating sequence (Garrido et al., 2004), and Rop1,
Table 2. Yeast Strains Used in This Study
Strain Relevant Genotype Reference
U. maydis
strains
FB1 a1 b1 Banuett and Herskowitz
(1989)
FB2 a2 b2 Banuett and Herskowitz
(1989)
FBD11 a1a2 b1b2 Banuett and Herskowitz
(1989)
FBD12-17 a2a2 b1b2 Banuett and Herskowitz
(1989)
FB1Dfuz7 a1 b1 Dfuz7 Mu
¨ller et al. (2003)
UME18 a1 b1 Dubc1 Garrido and Pe
´rez-Martı
´n
(2003)
UMP7 a1 b1 Dcru1 Castillo-Lluva et al.
(2004)
SG200 a1mfa2 bW2bE1 Bo
¨lker et al. (1995)
SONU8 a1 b1 P
crg
:fuz7
DD
This work
SONU24 a1 b1 Dadr1 Garrido and Pe
´rez-Martı
´n
(2003)
SONU27 a1 b1 prf1
nar
Garrido et al. (2004)
SONU34 a1 b1 P
crg
:fuz7
DD
Dprf1 This work
SONU38 a1 b1 prf1
nar
Dcru1 This work
SONU58 a1 b1 P
crg
:cln1-myc This work
SONU59 a1 b1 cln1-myc This work
SONU64 a1 b1 cln1
nar
This work
SONU68 a1a2 b1b2 cln1/Dcln1 This work
SONU73 a1 b1 cln1
crg
This work
SONU75 a2 b2 Dcln1 This work
SONU76 a1 b1 Dcln1 This work
SONU79 a1 b1 Dcln1 Dcru1 This work
SONU84 a1 b1 P
crg
:cln1-GFP This work
SONU115 a1 b1 prf1
nar
cln1
crg
This work
SONU130 a1mfa2 bW2bE1 Dcln1 This work
S. cerevisiae
strains
BF305-15#21 cln1THIS3 cln2TTRP1
GAL1-CLN3
Tyers et al. (1993)
SCS1 cln1THIS3 cln2TTRP1
GAL1-CLN3/p426-GPD
This work
SCS2 cln1THIS3 cln2TTRP1
GAL1-CLN3/pCM194
This work
SCS3 cln1THIS3 cln2TTRP1
GAL1-CLN3/pCLN1
This work
Table 3. Oligonucleotides Used in This Study
Primer Sequence
CLN1-KO1 59-CCCGGTACCGACACCATCGCTGCATGA-39
CLN1-KO2 59-CCGAATTCTTGAGATCGATGAGGGCCTTCCA-39
CLN1-KO3 59-CCCGAATTCTCTCTTTATTCATTGCAC-39
CLN1-KO4 59-CCCGGTACCGCCAGAATGCTAATCGAG-39
CLN1-N5 59-TCGCCATATGACCGCCCTCTGTCAGCTCGA-39
TGCLN1-3 59-GCGAATTCGAACTGCTGGACATTAACG-39
SCG1-1 59-GGACTAGTTTTCCACCCCATCTCAACG-39
SCG1-2 59-CGGAATTCGAGAGAGAAAGATGCTTGC-39
3556 The Plant Cell
which binds Rop1 recognition sites (Brefort et al., 2005). Whether
the Cdk1-cyclin complexes act inhibiting any of these regulatory
inputs or some new regulatory player is currently unknown, and
future experiments will address these issues.
Why in U. maydis are mating and mitosis incompatible op-
tions? We can imagine one possible explanation. U. maydis is
a saprophytic organism that in nature enters the sexual cycle only
in particular conditions (the surface of its host plant) that most
likely have a poor nutritional environment. Under favorable
nutritional conditions, U. maydis cells would prefer to reproduce
asexually by means of the mitotic cell cycle. An active mitotic
cycle correlates with high Cdk1-cyclin activity. By contrast, when
they experience starvation, for instance in the plant surface, the
cell cycle is turned down; therefore, the window for mating
process begins with the induction of sexual development.
Therefore, it seems logical that smut cells need a system to
carefully time the start of sexual development, when nutrients
become limiting and the possibilities to proliferate by fast mitotic
divisions are uncertain. The presence of a control system in-
volving positive (cln1 and clb1) and negative (cru1-APC) regu-
lators of cell cycle progression may constitute a sophisticated
mechanism by which the optimal time for mating and, therefore,
virulence are determined. This kind of dichotomy could be more
general than expected in fungi. In S. cerevisiae, where mitosis
and sporulation are incompatible options, a negative crosstalk
takes place by the inhibitory effect of G1 cyclins on IME1
transcription, the master regulator of sporulation (Colomina
et al., 1999). Also, in S. pombe, Puc1 contributes to negative
regulation of the timing of sexual development and functions at
the transition between cycling and noncycling cells, although the
putative target of this inhibition is currently unknown (Forsburg
and Nurse, 1994).
Cln1 and Virulence
One of the aims of this work was to define the role of G1
regulators in U. maydis virulence. Previous studies (Castillo-
Lluva et al., 2004; Garcı
´a-Muse et al., 2004) indicated that in U.
maydis, an accurate control of the cell cycle is predicted to be
necessary not only for cells growing in axenic conditions but also
during the infection process. However, we found that a solo-
pathogenic strain lacking the cln1 gene was fully virulent,
suggesting a minor role, if any, of this regulator during the
pathogenic phase. It is possible that in spite of the dramatic
effect during the yeast phase, Cln1 doesn’t play a role during the
hyphal phase of life cycle in U. maydis, suggesting that the cell
cycle may not be regulated in the same manner in these two cell
types. This situation is reminiscent of C. albicans, where there is
a differential requirement for the G1 cyclin Cln3 in yeast versus
hyphal cells (Bachewich and Whiteway, 2005; Chapa y Lazo
et al., 2005).
Strikingly, we found that haploid cells were unable to infect
plants. However, since mutant solopathogenic strains were fully
virulent, we traced the deficiency to steps previous to the cell–
cell fusion process. Although we cannot discard a direct role of
Cln1 in this process, we believe that the observed defect is
a consequence of the severe morphogenetic deficit of cells
lacking Cln1. Haploid Dcln1 cells secreted less pheromone than
wild-type cells and had an impaired response to exogenous
pheromone. Conditions that affect a correct morphogenesis,
such as treatment of cells with the actin inhibitor latrunculin A
(Fuchs et al., 2005) or mutations in the myo5 gene, encoding
a class-V myosin (Weber et al., 2003), also result in defects
during early steps of mating, strengthening the importance of
a correct morphogenesis during the mating process.
METHODS
Strains and Gr owth Conditions
The yeast strains used in this study are listed in Table 2. Saccharomyces
cerevisiae cells were grown in defined minimal (synthetic complete)
media supplemented with the appropriate nutrients for plasmid selection
(Sherman et al., 1986), and the Ustilago maydis cells were grown in YEP-
based medium, CMD, or minimal medium (Holliday, 1974). The expres-
sion of genes under the control of the crg1 and nar1 promoters, FACS
analysis, and cell cycle arrest were all performed as described previously
(Brachmann et al., 2001; Garcı
´a-Muse et al., 2003, 2004).
DNA, RNA, and Protein Analysis
The procedures used here for U. maydis RNA isolation and RNA gel blot
analysis, for DNA isolation and transformation, for obtaining protein
extracts, for Suc1 purification of Cdk complexes, and for immunoprecip-
itation and protein gel blot analysis have all been described previously
(Tsukuda et al., 1988; Garrido and Pe
´rez-Martı
´n, 2003; Garcı
´a-Muse et al.,
2004). The anti-PSTAIRE (Santa Cruz Biotechnology) and anti-myc 9E10
(Roche Diagnostics) antibodies were diluted 1:10,000 in phosphate-
buffered saline þ0.1% Tween þ10% dry milk for use. Anti-mouse
Ig-horseradish peroxidase and anti-rabbit Ig-horseradish peroxidase
(Roche Diagnostics) were used as secondary antibodies at a dilution of
1:10,000. All protein gel blots were visualized using enhanced chemilu-
minescence (Renaissance; Perkin-Elmer).
Plasmid and Strain Constructions
The plasmids used in this study are described below, and the oligonu-
cleotides are shown in Table 3. The PCR fragments amplified were
sequenced with an automated sequencer (ABI 373A; Applied Biosys-
tems) and analyzed with standard bioinformatic tools. To generate the
different strains, the constructs indicated were used to transform proto-
plasts as previously described (Tsukuda et al., 1988). The integration of
the plasmids into the corresponding loci was verified by diagnostic PCR
and subsequently by DNA gel blotting.
The cln1 gene was deleted using the pKOCLN1 plasmid. This plasmid
was produced by ligating a pair of DNA fragments flanking the cln1 ORF
into pNEB-Cbx (þ), a U. maydis integration vector containing a carboxine
resistance cassette (Brachmann et al., 2001). The 59fragment spans from
nucleotide 2250 to nucleotide 321 (considering the adenine in the ATG
as nucleotide þ1) and was produced by PCR of U. maydis genomic DNA
using the primers CLN1-KO1 and CLN1-KO2. The 39fragment spans from
nucleotide þ1314 to nucleotide þ1666 and was produced by PCR
amplification using the primers CLN1-KO3 and CLN1-KO4. After di-
gestion with KpnI, the pKOCLN1 plasmid was integrated by homologous
recombination into the cln1 locus.
To overexpress the cln1 gene, the cln1 ORF was first tagged with three
copies of the myc epitope. A fragment NdeI-EcoRI carrying the entire cln1
ORF sequence without the stop codon was obtained by PCR amplifica-
tion of U. maydis genomic DNA with the primers CLN1-N5 and TGCLN1-3
and subsequent digestion with the appropriated enzymes. This fragment
G1 Cyclin Controls Mating in Smuts 3557
was cloned into the plasmidpGNB-myc, a pGEX-2T derivative (Pharmacia
Biosciences) that carries three copies of the myc epitope (Garrido et al.,
2004), permitting the expression of the tagged fusion protein in bacteria.
The cln1-myc fusion was excised from the resulting plasmid (pGNB-cln1-
myc) as a NdeI-AflII fragment and subcloned into the pRU11 plasmid
(Brachmann et al., 2001). The pRU11 plasmid is an integrative U. maydis
vector that contains the crg1 promoter (Brachmann et al., 2001, and the
resulting plasmid (pRU11-cln1-myc) was linearized and integrated into
the cbx1 locus by homologous recombination.
To overexpress the cln1-GFP fusion, the plasmid pRU11-cln1-myc was
digested with EcoRI, and a 1-kb EcoRI fragment from p123 (Spellig et al.,
1996) carrying the gfp ORF was cloned in phase to produce pRU11-cln1-
GFP, which was linearized and integrated into the cbx1 locus by
homologous recombination.
To produce the conditional cln1
nar
and cln1
crg
alleles, we ligated two
fragments into pRU2 and pRU11, these being U. maydis integration
vectors containing the promoter of the nar1 gene (Brachmann et al., 2001)
and the promoter of the crg1 gene (Bottin et al., 1996), respectively. The 59
fragment was produced by PCR using the primers CLN1-KO1 and
CLN1-KO2, while the 39fragment was isolated from pGNB-cln1-myc.
The resulting pCLN1nar and pCLN1crg plasmids were linearized and
integrated into the cln1 locus by homologous recombination.
To tag the endogenous cln1 locus, the plasmid pRU11-cln1-myc
was digested with AflII-SexAI to remove the P
crg1
promoter as well as
the 59half of the cln1 gene. The resulting plasmid, pCLN1-MYC, was
linearized with BstXI and integrated into the cln1 locus by homologous
recombination.
To express the cln1 gene in S. cerevisiae, we PCR amplified the cln1
ORF from U. maydis genomic DNA with the primers SCG1-1 and SCG1-2.
This PCR fragment was cloned into p426GPD, a centromeric plasmid
carrying the URA3 marker and the promoter of the GPD1 gene (Mumberg
et al., 1995). The plasmid pCM194 contains the CLN3 gene under its own
promoter (Gallego et al., 1997).
Microscopy
A Leica DMLB microscope with phase contrast was used to analyze the
material, coupled to a Leica 100 camera. Epifluorescence was observed
using standard fluorescein isothiocyanate and DAPI filter sets, and
images were processed with Photoshop (Adobe Systems). Analysis of
GFP fusions was done using a Nikon Eclipse 600 FN microscope coupled
to a Hamamatsu ORCA-100 cooled CCD camera that was controlled by
MetaMorph software (Universal Imaging). Nuclear staining was per-
formed with DAPI as described previously (Garcı
´a-Muse et al., 2003),
and WGA staining was performed as described by Castillo-Lluva et al.
(2004).
Mating and Plant Infection
To test for mating, compatible strains were cospotted on charcoal-
containing plates (Holliday, 1974), which were sealed with Parafilm and
incubated at 218C for 48 h.
Pheromone stimulation was performed as described previously (Weber
et al., 2003). Synthetic a2 pheromone (a gift of G. Steinberg) was used at
a final concentration of 2.5 ng/mL.
Plant infection was performed as described by Gillissen et al.
(1992) using the maize (Zea mays) cultivar Early Golden Bantam (Old
Seeds).
Sequence Analyses
Protein sequences of fungal G1 cyclins were downloaded from PubMed
(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi). Alignments (see Sup-
plemental Figure 1 online) were made with ClustalX (Thompson et al.,
1997). Phylogenetic dendrograms were constructed using NJPlot
(pbil.univ-lyon1.fr/software/njplot.html) with the minimum evolution or
maximum parsimony algorithm and gap deletion option, based on 500
replicates. Domain analysis was performed using Pfam and Paircoil
(www.expasy.ch). PEST sites were predicted by PESTfind (embl.bcc.
univie.ac.at/htbin/embnet/PESTfind).
Accession Numbers
U. maydis Cln1 was identified in the genomic sequence of U. maydis (http://
www.broad.mit.edu/annotation/fungi/ustilago_maydis/index.html) as a
hypothetical protein (um04791); the new accession number is
DQ017758. Other sequence data from this article can be found in the
GenBank/EMBL data libraries under the following accession numbers: S.
cerevisiae cyclins Cln1 (AAA65724), Cln2 (AAA65725), and Cln3
(CAA32143); S. pombe Puc1 (CAA18649); C. albicans Cln1 (CAA56336),
Cln3 (EAK96708), and Hgc1 (EAK99984). We also included in our analysis
the sequence of putative G1 cyclins from two basidiomycetes: Crypto-
coccus neoformans (Cnd06233) and Coprinus cinerea (ccd00715).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Alignment of the Cyclin Boxes of Charac-
terized G1 Cyclins from Fungi.
Supplemental Figure 2. Formation of Appressoria by Wild-Type and
Dcln1 Solopathogenic Strains.
Supplemental Figure 3. Cln1 Is Not Required for Hyperpolarized
Growth Induced by the Mating Program.
ACKNOWLEDGMENTS
We thank M.A. Pen
˜alva (Centro de Investigaciones Biolo
´gicas, Madrid,
Spain) for essential help with fluorescence microscopy, Gero Steinberg
(Max Planck Institute for Terrestrial Microbiology, Marburg, Germany)
for the valued gift of synthetic a2 pheromone, and Martı
´Aldea
(Universidad de Lerida, Spain) for S. cerevisiae strains. We wish to
thank the anonymous referees for excellent suggestions that clearly
improved the work. This work was supported by a grant from the
Ministerio de Ciencia y Tecnologı
´a (BIO2002-03503) and by a grant from
the Comunidad Auto
´noma de Madrid (07B/0040/2002). S.C.-L. was
a recipient of a Formacio
´n de Personal Investigador fellowship from the
Ministerio de Ciencia y Tecnologı
´a.
Received July 18, 2005; revised August 20, 2005; accepted October 5,
2005; published October 28, 2005.
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