Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco-UAM, 28049 Madrid, Spain
* Author for correspondence (e-mail: jperez{at}cnb.uam.es)
Accepted 13 May 2005
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Summary |
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Key words: Ustilago maydis, cell cycle, inhibitory phosphorylation, Wee1-like kinases, phytopathogenic fungus
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Introduction |
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In Saccharomyces cerevisiae, the budding yeast homolog of Wee1, Swe1, has been linked to a checkpoint that delays cell cycle progression in response to morphogenetic defects (Booher et al., 1993; Sia et al., 1996
). Swe1 also modulates pseudohyphal growth in a broad spectrum of conditions (La Valle and Wittenberg, 2001
; Martínez-Anaya et al., 2003
). A role for Swe1 in the control of cell size was suggested by the finding of a swe1 mutant allele in a screening for whi phenotype (Jorgersen et al., 2002) and more recently it was shown that Swe1 delays entry into mitosis and is required for the control of cell size (Harvey et al., 2003). The common role of the Wee1 and Swe1 kinases may also apply to the filamentous fungus Aspergillus nidulans, where the Wee1-like kinase AnkA controls septum formation (De Souza et al., 1999
; Kraus and Harris, 2001
). In A. nidulans cells, predivisional hyphae cannot septate until a specific cell size is attained, indicating that septum formation is coordinated with cell growth (Wolkow et al., 1996
).
Here, we set out to investigate the influence of inhibitory phosphorylation of the catalytic subunit of mitotic CDK on cell growth and pathogenicity of the corn smut fungus Ustilago maydis. This pathogen is perfectly suited to analyze the relationships between cell cycle, morphogenesis and pathogenicity (Basse and Steinberg, 2004). Haploid cells of this fungus are unicellular and divide by budding. Induction of the pathogenic phase requires the mating of two compatible haploid cells and after cell fusion, the generation of an infective dikaryotic filament that invades the plant (Kahmann et al., 2000
). The different morphological changes that the fungal cells undergo during the pathogenic process provide evidence of the tight control of the cell cycle in these transitions. Indeed, we recently showed that manipulation of the transcription of mitotic cyclins affects hyphal proliferation within the plant, resulting in fungal cells unable to produce a successful infection (García-Muse et al., 2004
). We also reported that mutations in the Fizzy-related APC activator Cru1 disrupted different stages of plant infection by U. maydis (Castillo-Lluva et al., 2004
). As a further step toward elucidating the role of cell cycle regulation in the virulence of this fungus, we have analyzed whether impairing the inhibitory phosphorylation of CDK affects the ability of U. maydis to infect plants. To achieve this, we took advantage of cells expressing a constitutively unphosphorylated cdk1 allele to show that deficient inhibitory phosphorylation affects morphogenesis, the coordination of cell growth with the cell cycle, the S phase checkpoint control and virulence. We also characterized the U. maydis Wee1 kinase ortholog and found that Wee1 is required to control the G2/M transition in U. maydis. The data reported here reinforce the proposed connection between the cell cycle, morphogenesis and pathogenicity in U. maydis.
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Materials and Methods |
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DNA, RNA and protein analysis
U. maydis DNA and RNA isolation, the preparation of protein extracts, northern and western blotting, and immunoprecipitations were all performed as described previously (Tsukuda et al., 1988; Garrido and Pérez-Martín, 2003
; García-Muse et al., 2004
; Garrido et al., 2004
). The anti-PSTAIRE (Santa Cruz Biotechnology), anti-myc 9E10, and anti-VSV (Roche Diagnostics Gmb) antibodies were all used at a dilution of 1:10,000 in phosphate-buffered saline + 0.1% Tween + 10% dry milk. The anti-phospho-Cdc2 (Tyr15) antibody (Cell Signaling) was used according to the manufacturer's instructions. The anti-mouse Ig horseradish peroxidase and anti-rabbit Ig horseradish peroxidase (Roche Diagnostics Gmb) secondary antibodies were used at a dilution of 1:10,000. All western blots were visualized by enhanced chemiluminescence (Renaissance®; Perkin Elmer).
Plasmids and constructs
Plasmid pGEM-T easy (Promega) was used for cloning, subcloning and sequencing of genomic and PCR fragments. The plasmids pRU11, pRU2, pCU2 and pCU3 were used to express genes under the control of Pcrg1, Pnar1, Pscp and Ptef1 promoters, respectively, and the plasmids pBS-MYC-HYG and pGNB-myc to produce C-terminal myc-tagged protein fusions as already described (Brachmann et al., 2001; Brachmann, 2001
; García-Muse et al., 2004
; Garrido et al., 2004
). The oligonucleotides used for PCR amplification are shown in Table 2 and the PCR fragments generated were analyzed using an automated sequencer (ABI 373A) and standard bioinformatic tools. To construct the different U. maydis strains, protoplasts were transformed with the constructs indicated using the protocol described by Tsukuda et al. (Tsukuda et al., 1988
). Integration of the plasmids into the corresponding loci was verified by diagnostic PCR and Southern blot analysis in all cases.
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The mutant cdk1AF allele was constructed by assembling two PCR fragments that carried the T14A and Y15F mutations, generated with the primer pairs CDK1/CDK2-b and CDK3-b/CDK4. The wild-type cdk1 allele was amplified using the CDK1 and CDK4 primers. In both cases, the resulting 1.1 kb fragment was inserted into pGNB-myc to obtain the myc-epitope-tagged protein. The tagged alleles were then inserted into pCU3 (Ptef1-dependent expression) or pRU2 (Pnar1-dependent expression) to construct the pCU3-Cdk1, pCU3-Cdk1AF, pRU2-Cdk1 and pRU2-Cdk1AF plasmids. These plasmids were linearized and integrated into the cbx1 locus by homologous recombination, as described by Brachmann et al. (Brachmann et al., 2001). To express the cdk1 and cdk1AF alleles under the control of the plant-specific promoter Pmig1, a 2 kb fragment carrying the Pmig1 promoter was inserted into pCU3-Cdk1 and pCU3-Cdk1AF to replace a 0.25 kb fragment carrying the Ptef1 promoter. This fragment was obtained by PCR amplification with primers MIG1-1 and MIG1-2, using U. maydis genomic DNA as the template. The resulting plasmids, pMIG1-Cdk1 and pMIG1-Cdk1AF were integrated by homologous recombination into the mig1 locus.
To produce a C-terminal tagged version of Wee1, a 4.5 kb fragment encompassing the wee1 ORF without the stop codon was amplified by PCR from U. maydis genomic DNA with the primers WEE1-32 and WEE1-31. This fragment was cloned into pGEM-T easy to produce the pGEMT-Wee1 plasmid. A 2.6 kb fragment from pGEMT-Wee1 was inserted into pBS-MYC-HYG to produce the endogenous myc-tagged wee1, and the resulting pBS-Wee1-myc plasmid was integrated into the wee1 locus by homologous recombination. To overexpress wee1, a C-terminal myc-epitope tag was first introduced by inserting the 4.5 kb pGEMT-Wee1 fragment into pGNB-myc. From the resulting pGNB-wee1-myc plasmid, a 4.7 kb fragment carrying the tagged allele was inserted into the pRU11 vector (Pcrg1-dependent expression). The resulting pRU11-Wee1-myc plasmid was linearized and integrated into the cbx1 locus by homologous recombination as described by Brachmann et al. (Brachmann et al., 2001).
To delete the wee1 gene, we used the pKOWee1 plasmid generated by ligating two DNA fragments flanking the wee1 ORF into pNEB-HYG (+), a U. maydis integration vector containing a hygromycin resistance cassette (Brachmann et al., 2001). The 5' fragment was produced by PCR with the primers WEE1-7 and WEE1-8 using U. maydis genomic DNA as the template, and the 3' fragment using the primers WEE1-9 and WEE1-10. After linearization, the pKOWee1 plasmid was integrated into the wee1 locus by homologous recombination.
To produce a conditional wee1nar allele we inserted two PCR fragments into pRU2. The 5' fragment was produced using the primers WEE1-35 and WEE1-36 and spanned from nucleotide 872 to nucleotide 120 (considering the adenine in the ATG as nucleotide +1). The 3' fragment was obtained with the primers WEE1-32 and WEE1-27 and spans from nucleotide +1 to nucleotide +1524. The resulting plasmid pWEE1nar was linearized and integrated into the wee1 locus by homologous recombination.
To produce the wee1scp allele, a 2.28 kb fragment from pWEE1nar was inserted into pCU2 (Pscp-dependent expression). The resulting plasmid pWEE1scp was linearized and integrated into the wee1 locus by homologous recombination.
Microscopy
Microscopy was carried out using a Leica DMLB microscope with phase contrast optics. Standard FITC and DAPI filter sets were used for epifluorescence analysis of nuclear staining with DAPI (see García-Muse et al., 2003) and WGA staining, performed as described by Castillo-Lluva et al. (Castillo-Lluva et al., 2004
). Photomicrographs were obtained with a Leika 100 camera and the images were processed with Photoshop (Adobe).
Mating and plant infection
To test for mating, compatible strains were co-spotted on charcoal-containing PD plates that were sealed with parafilm and incubated at 21°C for 48 hours (Holliday, 1974). Plant infections were performed with the maize cultivar Early Golden Bantam as described previously (Old Seeds, Madison, WI, USA) (Gillissen et al., 1992
). Filaments inside the plant tissue were stained with Chlorazole Black E as described by Brachmann et al. (Brachmann et al., 2003
).
Sequence analyses
Protein sequences of fungal Wee1-like kinases were downloaded from PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi). Alignments and phylogenetic dendrograms were constructed using ClustalW and NJPlot programs (Thompson et al., 1997).
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Results |
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S phase checkpoint control in U. maydis could be mediated through inhibitory phosphorylation of Cdk1
Inhibitory phosphorylation of the catalytic subunit of CDK is part of the S phase checkpoint control in response to damage or incompletely replicated DNA in several organisms. For instance, S. pombe, A. nidulans and human cells are dependent of this regulation. In contrast, S. cerevisiae cells do not require inhibitory phosphorylation to arrest cell cycle after activation of a S phase checkpoint (Lew and Kornbluth, 1996). To address whether in U. maydis the inhibitory phosphorylation is involved in the response to S phase checkpoint, we firstly examined the levels of Tyr15 phosphorylation in response to an arrest in S phase caused by addition of 1 mg/ml hydroxyurea (García-Muse et al., 2004
). We found that the level of tyrosine-phosphorylated Cdk1 increased upon addition of hydroxyurea (HU) (Fig. 2A), suggesting an involvement of this negative regulation in the S phase checkpoint as in other organisms. To reinforce this conclusion, we tested cells expressing high levels of the unphosphorylated cdk1AF allele for sensitivity to HU at sub-lethal concentrations. As control we used wild-type cells and cells expressing high levels of wild-type cdk1 alelle. Consistently, we found that cells with impaired inhibitory phosphorylation showed a high degree of HU sensitivity (Fig. 2B).
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We created a fungal strain in which the genomic copy of wee1 was replaced with a C-terminal myc-tagged Wee1 protein. Cell growth and morphology was not noticeably affected by this replacement (not shown). Using this strain, we investigated whether the levels of Wee1 fluctuated in different phases of the cell cycle. Because no reproducible synchronization method is so far available for U. maydis, we performed western blotting on protein extracts isolated from cultures of wild-type cells enriched in G1 phase, or cells arrested in S phase by the presence of hydroxyurea, or M phase with benomyl. We detected Wee1 under all conditions, but it appeared to undergo a mild down-regulation that coincided with M phase arrest (Fig. 3D), as described for Swe1 in S. cerevisiae (Harvey and Kellogg, 2003) and Wee1 in S. pombe (Aligue et al., 1997
). We also analyzed the mRNA levels and found that wee1 mRNA is preferentially expressed at G1 phase with the lowest levels again being found in M phase-arrested cells (Fig. 3E), as described for SWE1 in S. cerevisiae (Rey et al., 1996
).
High levels of wee1 expression causes cell cycle arrest at G2 phase
Overexpression of Wee1-related kinases delays entry into mitosis in several systems, resulting in G2 arrest as a consequence of accumulation of phosphorylated inactive CDK complexes (Russell and Nurse, 1987; Booher et al., 1993
; McGowan and Russell, 1995
). To determine whether overexpression of wee1 has similar inhibitory activity in U. maydis, an extra copy of wee1 was introduced into wild-type U. maydis cells. This additional wee1 allele was under the control of the crg1 promoter which can be induced by arabinose and repressed by glucose (Bottin et al., 1996
). These cells were unable to grow in solid medium containing arabinose (Fig. 4A) indicating that the overexpression of wee1 was deleterious to the cells.
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In summary, high levels of Wee1 induce G2 arrest, correlated with a high level of Tyr15 phosphorylation of Cdk1 in U. maydis. On these grounds, we consider U. maydis Wee1 to be a Wee1-related protein kinase.
Wee1 is essential for the survival of U. maydis
To further analyze the function of Wee1, we inactivated one wee1 allele in the diploid FBD11 strain, replacing it with a hygromycin-resistance cassette, generating the wee1 null allele. When the meiotic progeny of this strain were analyzed after sporulation, no hygromycin-resistant cells were found, indicating that wee1 is an essential gene. Therefore, we constructed another strain in which the Wee1 protein could be conditionally ablated. This was achieved using the U. maydis nar1 promoter that is induced by growing the cells in nitrate as the nitrogen source, and strongly repressed when the cells are grown in rich medium (YPD) (Brachmann et al., 2001
). A chimeric allele (wee1nar) was constructed by fusing the nar1 promoter to the coding region of wee1 and the native allele was replaced by this conditional allele. In the conditional mutant, a three-fold increase was observed in wee1 mRNA with respect to the levels in wild-type cells in permissive (minimal medium containing nitrate, MMNO3) but not in restrictive conditions (YPD medium; Fig. 5A). Conditional mutant cells grew on solid medium containing nitrate at a rate similar to control wild-type cells. However, in accordance with the essential role of the Wee1, they were unable to form colonies when shifted to solid YPD medium (Fig. 5B).
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We analyzed the growth of this conditional strain in liquid medium. In restrictive conditions the conditional cells generated chains of rounded cells that divided by septation (Fig. 6). The cell compartments were shorter than normal (6 µm on average versus 17 µm in wild-type U. maydis cells), and contained at least one nucleus. Nevertheless, when the DNA content was analyzed by FACS we found that in restrictive conditions, wee1nar cells accumulated DNA in genome sized multiples (Fig. 4D). Cell analysis using microdensitometry (Snetselaar and McCann, 1997) indicated that the relative intensity of nuclei fluctuate between two peaks, presumably corresponding to 1C and 2C DNA content (not shown), which is consistent with normal DNA replication. Accordingly, these cell aggregates were reminiscent of those seen in cells expressing the constitutively unphosphorylated cdk1AF allele. Our interpretation is that impairing the inhibitory phosphorylation of Cdk1 promoted premature entry into mitosis, resulting in the inability to produce a bud and followed by division through septation.
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To correlate Wee1 function with inhibitory phosphorylation in Cdk1, wild-type and conditional cells were grown in permissive conditions until the mid-exponential phase and then the cultures were shifted to restrictive conditions. Samples were collected at different times and the degree of Tyr15 phosphorylation was analyzed by Western blotting with the anti-Cdc2-Y15P antibody. A dramatic reduction in the levels of Tyr15 phosphorylation was clearly evident in extracts obtained from conditional cells growing in restrictive conditions (Fig. 5C).
Taken together these results support the notion that Wee1-mediated inhibitory phosphorylation of Cdk1 controls entry into mitosis and hence the length of the G2 phase in U. maydis.
Wee1 controls the Cdk1-Clb2 complex
We previously demonstrated the specificity of the B-type cyclin Clb2 to the G2/M transition where it appears to be rate-limiting for entry into mitosis in U. maydis (García-Muse et al., 2004). Cell growth was arrested in G2 phase when Clb2 function was impaired, these cells developing an elongated bud that displayed active polarized growth, a phenotype resembling that generated by wee1 overexpression. In contrast, high levels of clb2 expression resulted in short cells that divide by septation, a phenotype reminiscent of cells that do not express wee1 or in which a constitutively unphosphorylated cdk1AF allele was overexpressed. Together, these observations suggest that the Cdk1-Clb2 complex could be a target of Wee1.
To evaluate whether Wee1 antagonizes the Cdk1-Clb2 complex, we generated a double conditional mutant wee1nar clb2nar. When we compared this mutant with the respective single wee1nar and clb2nar conditional mutants in restrictive conditions, the concurrent down-regulation of wee1 and clb2 transcription produced a phenotype identical to that caused by the down-regulation of clb2 expression alone (Fig. 7A). This epistastic relationship located clb2 genetically downstream of wee1. We then analyzed whether high levels of Wee1 could overcome the effects of high levels of Clb2 on cell morphology. Cells with high constitutive levels of clb2 expression (replacing the native clb2 promoter with the strong constitutive hsp70 promoter) and carrying an ectopic arabinose-inducible wee1 allele expressed the phenotype of cells overexpressing clb2 when wee1 expression was not induced (YPD, Fig. 7B). However, when these cells were transferred to arabinose-containing medium, the cells elongated, resembling wild-type cells that overexpress wee1 (Fig. 7B, YPA). This result provides further evidence of antagonism between clb2 and wee1.
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Transcriptional regulation of wee1 is important to adapt cell size to different nutritional conditions
Fungal cells adjust their cell cycle depending on environmental conditions. For instance, in response to poor nutritional conditions, this adjustment results in an enlargement of the generation time avoiding the generation of abnormally small daughter cells (Rupes, 2002). In U. maydis, this enlargement takes place by increasing the length of the G1 phase until the cell reaches a minimum size to enter a new S phase (Snetselaar and McCann, 1997
). We have previously shown that the APC adaptor Cru1 is important in U. maydis to determine the length of G1 and cell size in response to nutritional conditions. Indeed, the mRNA levels of cru1 are regulated by the quality of the growth medium (Castillo-Lluva et al., 2004
). In this sense, cell size control in U. maydis seems to be more similar to S. cerevisiae, in which it takes place mainly in G1, than to fission yeast, in which G2/M transition is the primary cell size control point (Rupes, 2002
). However, the above results showed that the levels of Wee1 seem to be important in determining the length of the G2 phase and hence cell size. Therefore, to examine whether Wee1 has a role in coordinating cell growth, cell division and nutritional conditions in U. maydis, we first addressed whether the levels of wee1 mRNA were affected by the growth medium. Total RNA was extracted from wild-type cells cultured in YPD, CMD and MMD, and the levels of wee1 mRNA analyzed. It was readily apparent that wee1 mRNA levels increased as the quality of the medium decreased (Fig. 8A).
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How important is wee1 transcriptional regulation for cells to adapt to different nutritional conditions? To address this question we exchanged the native wee1 promoter with the scp promoter, a weak U. maydis constitutive promoter that produces low levels of expression (Bölker et al., 1995a). The abundance of the mRNA produced by this promoter did not vary as a function of the nutritional conditions, and was similar to the wee1 levels produced in wild-type cells growing in rich medium (YPD, Fig. 8A). Moreover, the cells carrying the constitutive wee1scp allele were smaller than wild-type cells (Fig. 8B), and this difference was more obvious in less nutritional medium. This result suggests that up-regulation of Wee1 levels play some role in the adjustment of cell cycle in response to nutritional conditions, particularly in less favorable medium. Analysis of the FACS profile of wild-type and mutant wee1scp cells grown in the different media (Fig. 8C) showed an increase in the number of cells with 1C DNA content (i.e. cells in G1 phase). This bias towards cells in G1, suggests that the G1 period must be lengthened in these small cells until the minimum size required for overriding the Start-restraint is attained One possible interpretation of these results is that U. maydis uses two different cell size controls at the same time in response to nutritional conditions: one in G1 phase (related with the APC-Cru1 complex) and other in G2/M (related with the Wee1 levels), and that the absence of one of these controls must be compensated by the other. To reinforce this interpretation, we tried unsuccessfully to delete the cru1 gene in cells carrying the wee1scp allele. To determine if the deletion of the cru1 gene in wee1scp cells was lethal, we crossed haploid
cru1 (UMP9, marked with a hygromycin-resistance gene) cells with compatible haploid wee1scp cells (UMC44, marked with a carboxine resistance gene) and afterwards we analyzed the meiotic progeny. From 100 analyzed resulting haploid cells, none was found to carry the two mutant alleles, indicating a synthetic lethality.
Inhibitory phosphorylation could be required for polar growth during the infection process
Several results suggested an involvement of the inhibitory phosphorylation of Cdk1 in the maintenance of polarized growth in U. maydis. The conditional depletion of Wee1 or the expression of the cdk1AF allele generated cell aggregates composed of cells that often lost polarity and became almost spherical. In contrast, the overexpression of wee1 generated a highly polarized growth. The initiation of pathogenic development in U. maydis involves the activation of a strong polarized growth, as yeast-like budding cells switch, after mating, to tip-growing hyphae, so-called infective tubes (Kahmann et al., 2000). Therefore, we were interested in determining whether cells deficient in inhibitory Cdk1 phosphorylation were able to produce the infective tube. Since wee1 is essential in U. maydis, we took advantage of the cells expressing the cdk1AF allele to mimic defective inhibitory phosphorylation. The induction of infective tube formation can be easily scored by co-spotting compatible strains (i.e. with different a and b loci) on solid medium containing charcoal. On these plates, cell fusion and the development of an infective dikaryotic filament results in the formation of a white layer of aerial hyphae on the surface of the growing colony [Fuz+ phenotype (Holliday, 1974
)]. We generated compatible strains that expressed an ectopic copy of the cdk1AF allele under the control of the nar1 promoter. Mixtures of compatible wild-type and mutant strains were spotted on charcoal-containing minimal medium with ammonium (non-inducing conditions) or nitrate (inducing conditions) as a nitrogen source. Mixtures of wild-type cells produced a clear Fuz+ phenotype in both conditions. In contrast, when compatible strains carrying the cdk1AF allele were spotted or mixed with wild-type compatible strains, the Fuz+ phenotype did not develop (Fig. 9A). Furthermore, microscopic observation of the mutant mixtures growing on nitrate-containing charcoal plates confirmed the absence of dikaryotic hyphae (not shown). These results indicated that inhibitory phosphorylation is required for the formation of the dikaryotic infective filament. However, dikaryotic hyphal formation requires the fusion of compatible cells, and we therefore considered it possible that this phenotype is a secondary consequence of a defect in mating. To circumvent the need for cell fusion we generated the respective mutations in the solo-pathogenic strain SG200 (Bölker et al., 1995b
). This strain, which carries the information of the two mating types, is able to form infective tubes without a mating partner. Consistently with the charcoal assay, SG200 cells expressing the cdk1AF allele were unable to produce the infective filament (Fig. 9B).
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Taken together, these results strongly suggest that inhibitory phosphorylation of Cdk1 plays an important role at different points of the infection process and that this role could be related with the ability to support a sustained polarized growth.
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Discussion |
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The fact that wee1 overexpression causes arrest of U. maydis cells in G2 suggests that the Cdk1-cyclin complexes targeted by Wee1 are required for the G2/M transition. In U. maydis, the G2/M transition requires the participation of both the Cdk1-Clb1 and the Cdk1-Clb2 complexes (García-Muse et al., 2004). Accordingly, we have shown that the phosphorylated Cdk1 is associated to these B-type cyclins. The Tyr15 phosphorylated Cdk1 cannot be found in complexes with Cln1, indicating that as in other organisms, different cyclins target CDKs for distinct negative regulatory controls in U. maydis (Devault et al., 1992
; Booher et al., 1993
; Watanabe et al., 1995
). We believe that the primary target for Wee1 is the Cdk1-Clb2 complex. While Cdk1-Clb1 is required for the G1/S and G2/M transitions, Cdk1-Clb2 appears to be specific for G2/M transition and it seems to be a rate-limiting for entry into mitosis (García-Muse et al., 2004
). Indeed, clb2 overexpression produces a phenotype that resembles those produced by overexpression of the cdk1AF allele or by downregulation of wee1. In contrast, overexpression of clb1 produces lethal chromosome missegregation (García-Muse et al., 2004
). Furthermore, we found that high levels of Wee1 overcome the effects on cellular morphology imposed by high levels of Clb2. In contrast, the toxic effect of high levels of Clb1 (García-Muse et al., 2004
) cannot be suppressed by an abundance of Wee1 (J.P.-M., unpublished). However, since the Cdk1-Clb1 complex is also subjected to Tyr15 phosphorylation (albeit apparently less than the Cdk1-Clb2 complexes), we believe a fraction of the Cdk1-Clb1 complexes involved in the G2/M transition, are also targeted by the Wee1 kinase. As such, we assume that elements specific to the G1/S transition modify the capacity of the Wee1 kinase to phosphorylate the Cdk1-Clb1 complex.
Inhibitory phosphorylation seems to be essential for cell cycle regulation in U. maydis. This is similar to S. pombe in which two related kinases (Wee1 and Mik1) phosphorylate Cdc2, the deletion of either gene being lethal (Lundgren et al., 1991). In contrast, the gene encoding the Wee1-like kinase in S. cerevisiae (SWE1) is dispensable for growth (Booher et al., 1993
). The requirement in U. maydis for wee1 reflects the importance of controlling G2 phase length in this organism. Once DNA replication has occurred, U. maydis cells must decide whether to bud or to enter into a mating program, a decision that is taken in response to external stimuli (García-Muse et al., 2003
). Controlling the length of G2 seems to be primordial for U. maydis to make the correct decision. In addition, our data are in accordance with an interesting model proposed by Kellog (Kellog, 2003), which suggests that Wee1-related kinases monitor the total amount of polar growth that occurs. Fission yeast and U. maydis are rod-shaped cells and all growth occurs at the ends of the cell (polar growth). In contrast, S. cerevisiae cells undergo a brief period of polar growth during bud emergence, but then growth occurs over the entire surface of the bud (isotropic growth). In agreement with Kellog (Kellog, 2003), the loss of wee1 function causes a more severe phenotype in U. maydis than in S. cerevisiae, despite the fact that both organisms divide by budding. We believe that this may be because U. maydis relies almost entirely on polar growth that occurs during G2, whereas budding yeast only undergo a brief period of polar growth and then switch to isotropic growth.
Also, again in contrast with S. cerevisiae, two observations reported here also suggest that inhibitory phosphorylation of Cdk1 could be involved in the modulation of a S phase checkpoint response. We found that the amount of Tyr15 phosphorylation increases after treatment with HU, and that cells expressing the cdk1AF allele were hypersensitive to sub-lethal concentrations of HU (i.e. the S phase checkpoint defect is presumably the cause of the HU hypersensitivity).
Progression through the cell cycle is regulated principally before the onset of S phase and of mitosis. In both cases, a critical cell mass must be attained before progression occurs (Nurse, 1975; Fantes, 1977
). Recently, we described that in U. maydis, the absence of the APC adaptor Cru1 resulted in the inability to adjust G1 phase and hence in smaller than normal cells (Castillo-Lluva et al., 2004
). Here, our results show that there is a close relationship between wee1 expression and cell size. We generated two different wee1 alleles, wee1nar and wee1scp, that are transcribed more or less than the wild-type allele. Altering the expression of wee1 produced cells that were larger and smaller than normal. Wild-type cells can adjust their size to the exterior environment, such as the availability of nutrients (Fantes and Nurse, 1978). Thus, by regulating Wee1 function, growth signals could conceivably produce such an effect. We found that wee1 mRNA levels increased as the quality of the medium decreased. In poor medium cells spend longer in G1 phase than in rich medium (Castillo-Lluva et al., 2004
) and wee1 seems to be expressed preferentially in G1 phase. Hence, the differences in mRNA levels imposed by the growth medium could be easily explained without having to invoke mechanisms by which wee1 transcription is directly controlled by the nutritional conditions. Nevertheless, this latter possibility cannot be discarded.
Although minimal medium has been shown to extend the G1 phase of the U. maydis cell cycle (Castillo-Lluva et al., 2004), we propose that poorer medium may also delay progression to G2, and that this delay is dependent on Tyr15 phosphorylation. Such coordination in the length of the G1 and G2 phases is not only useful in conditions of limited nutrition but also to compensate for defects in each phase. For instance, G1 phase is not appreciable in cells carrying the wee1nar allele, which leads to a delay in mitosis and oversized buds (compare the FACS profile of wee1nar cells with wild-type cells growing in MM-NO3, Fig. 4B). In contrast, in wee1scp cells, which have low levels of wee1 mRNA and are small, there is a clear increase in the time spent by the cell in G1 phase. Furthermore, Cru1 is indispensable for the growth of wee1scp cells, probably because these double mutants cannot lengthen the G1 period to restrain S phase until the critical size to override the Start control is attained.
In addition to the influence on cell size, the cdk1AF allele or the down-regulation of wee1 had a marked effect on septation and cell pattern formation during vegetative growth. This indicates that septation in axenic cultures of U. maydis is normally prevented through a mechanism involving tyrosine phosphorylation of Cdk1. Indeed, septation in A. nidulans is regulated via tyrosine phosphorylation of nimXcdc2 (Harris and Kraus, 1998; Kraus and Harris, 2001
). Another possibility is that the failure to inhibit Cdk1 through phosphorylation impairs the coordination between cell cycle regulation and the transition to the budding mode of growth. In S. cerevisiae a checkpoint system monitors the emergence of the bud. This morphogenetic checkpoint coordinates bud emergence with the cell cycle through the activity of the Swe1 protein kinase and tyrosine phosphorylation of Cdc28 (Lew and Reed, 1995
; Sia et al., 1996
). This system is linked to the organization of the cytoskeleton and septin function through a protein kinase cascade that eventually inhibits Cdc28 by tyrosine phosphorylation (Carroll et al., 1998
; Longtine et al., 1998
; Barral et al., 1999
). If such a system were operative in U. maydis, it may become critical in axenic cultures where U. maydis cells grow by budding. The failure to inhibit Cdk1 by phosphorylation may prevent the normal synchronization and eventually contribute to the developmental defects we have observed.
One of the aims of this work was to define the interaction between the inhibitory phosphorylation of the catalytic subunit of mitotic CDKs and the program of virulence in U. maydis. We found that the expression of a constitutively unphosphorylated Cdk1 abolishes the ability of U. maydis to produce infective filaments. This inability could be explained assuming a role of Wee1 protein in the regulation of polarized growth. Alternatively, since the infective filament is cell cycle arrested in G2 phase (C.S. and J.P.-M., unpublished), it is tempting to speculate that G2 cell cycle arrest is mediated by the inhibition of Cdk1 by phosphorylation, and that the failure to induce arrest of the cell cycle impairs the formation of the infective tube. This interesting possibility will be clarified in future studies. By restricting the expression of the constitutively unphosphorylated cdk1 allele to fungi growing inside the plant, we bypassed the requirement of inhibitory phosphorylation for plant penetration. Hence, we were able to show that Tyr15 phosphorylation is also required for infection to progress inside the plant, since the symptoms of mutant cell infection do not progress beyond chlorosis. Interestingly, fungal cells expressing high levels of clb2 (García-Muse et al., 2004) showed a similar infection defect. Taken together, these data indicate that the accurate control of G2/M transition seems to be important for successful infection by U. maydis. It is worth bearing in mind that U. maydis cells grow in yeast-like unicellular form in saprophytic conditions. The induction of the pathogenic phase requires two compatible haploid cells to fuse and the generation of an infective dikaryotic filament that invades and proliferates inside the plant (Kahmann et al., 2000
). Studies of pseudohyphal development in S. cerevisiae have revealed the importance of controlling the G2/M transition for the production of the filamentous growth (for a review, see Rua et al., 2001
).
In summary, the results presented here reinforce the connections between the cell cycle and the induction of the pathogenic program in U. maydis. Moreover, these data highlight the potential significance of cell cycle regulation in microbial pathogenesis.
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