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First published online 26 July 2005
doi: 10.1242/jcs.02499

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Inhibitory phosphorylation of a mitotic cyclin-dependent kinase regulates the morphogenesis, cell size and virulence of the smut fungus Ustilago maydis

Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco-UAM, 28049 Madrid, Spain

View larger version (63K): [in a new window]   Fig. 1. The absence of inhibitory Cdk1 tyrosine phosphorylation causes severe morphological defects in U. maydis. (A) Cdk1 is phosphorylated at Tyr15. Protein extracts from wild-type and TAU17 cells expressing a FLAG-tagged version of Cdk1 (cdk1-1) were subjected to immunoblotting with anti-phospho-Cdc2 (Tyr15) and anti-PSTAIRE antibodies. Cdk2 is the U. maydis homolog of S. cerevisiae Pho85, which is also recognized by the anti-PSTAIRE antibody. (B) Expression of the ectopic myc-tagged Cdk1. Protein extracts from wild-type, and UMC5 and UMC6 cells expressing myc-tagged Cdk1 (Ptef1:cdk1-myc) and the constitutively unphosphorylated Cdk1 allele (Ptef1:cdk1AF-myc), respectively, were immunoblotted with anti-PSTAIRE antibodies. (C) UMC5 and UMC6 cells were grown to mid-exponential phase in rich medium (YPD) and then stained with DAPI and FITC-WGA, to visualize nuclei and cell walls, respectively. Scale bars: 20 µm.

View larger version (62K): [in a new window]   Fig. 2. Cdk1 phosphorylation and S phase checkpoint. (A) Effects of inhibition of DNA synthesis by hydroxyurea on Cdk1 phosphorylation. HU (1 mg/ml) was added to an early log phase culture of FB1 cells and samples were taken at 30 and 60 minutes after HU addition. (B) Impaired tyrosine phosphorylation of Cdk1 causes sensitivity to HU. Wild-type (FB1), and UMC5 and UMC6 cells expressing myc-tagged Cdk1 (Ptef1:cdk1-myc) and the constitutively unphosphorylated Cdk1 allele (Ptef1:cdk1AF-myc), respectively, were spotted as serial dilutions onto YPD plates containing a series of HU concentrations and the plates were incubated at 28°C for 3 days.

View larger version (56K): [in a new window]   Fig. 3. Sequence analysis of a U. maydis Wee1-like protein. (A) Schematic representation of the Wee1 protein in relation to other fungal Wee1-like proteins. The catalytic domains are shown in black and were identified using the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de). The percentages inside each box represent the sequence identity when compared to the U. maydis sequence. (B) Dendrogram of Wee1-like proteins. The tree was reconstructed using the ClustalW method (http://www.ebi.ac.uk/clustalw/). Bar: 0.05 substitutions per aa. (C) Comparison of the catalytic domain of U. maydis Wee1 kinase with that of related Wee1-like proteins. The roman numerals indicate the catalytic subdomains as designated by Hanks (Hanks, 1991). The shaded residues are amino acids characteristic of Wee1 family kinases. (D) Protein levels at different stages of the cell cycle. Extracts from UMC38 cells carrying a myc-tagged copy of Wee1 and arrested at S or M phase, G1 phase enriched or cells growing asynchronously (As) were immunoblotted. The same filters were probed with anti-myc and anti-PSTAIRE antibodies and Cdk1 levels were used as loading controls. (E) Levels of wee1 expression at different cell cycle stages. RNA extracted from wild-type FB1 cells arrested at S phase or M phase, or enriched in G1 phase and growing asynchronously (As), was analyzed by northern blotting. The filter was hybridized with probes for wee1 and 18s rRNA as a control of loading.

View larger version (56K): [in a new window]   Fig. 4. Ectopic expression of wee1. (A) The growth of cells expressing an ectopic copy of the wee1 gene was examined by spotting serial dilutions of exponential cultures of FB1 (wild type) and UMC33 (Pcrg1:wee1) strains in solid rich medium with either 2% glucose (YPD, non-inducing conditions) or 2% arabinose (YPA, inducing conditions). Plates were incubated for 3 days at 28°C. (B) Micrographs showing the cell morphology of FB1 and UMC33 cells after 6 hours of growth in YPA liquid cultures (inducing conditions). Note the elongated shape and the presence of a single nucleus (DAPI staining). Scale bars: 20 µm. (C) Microtubule network of UMC41 cells, carrying an -tubulin-GFP fusion and expressing high levels of wee1 (GFP-Tub1 epifluorescence; Scale bar: 25 µm. (D) FACS analysis of cell DNA content of FB1 and UMC33 in non-inducing (YPD) and inducing conditions (YPA). Samples were removed 0, 2, 4 and 6 hours after transfer to conditional medium. The shift to a DNA content higher than 2C observed in UMC33 cells incubated in CMA for 6 hours was due to mitochondrial DNA staining. (E) Western analysis of inhibitory phosphorylation after wee1 overexpression. Protein extracts from the FB1 and UMC33 cultures incubated in inducing conditions for the times indicated were obtained (YPA; in hours). The overexpressed wee1 allele was myc-tagged and detected with an anti-MYC antibody. Cdk1 was visualized with anti-phospho-Cdc2 (Tyr15) and anti-PSTAIRE antibodies.

View larger version (67K): [in a new window]   Fig. 5. Conditional removal of Wee1. (A) Levels of wee1 mRNA in the conditional strain. The wild-type FB1 and conditional UMC23 (wee1nar) strain were grown for 8 hours in permissive (minimal medium with nitrate, MMNO3) or restrictive conditions (rich medium, YPD). The RNA was extracted and analyzed by northern blotting, loading 10 µg total RNA per lane. 18s rRNA was used to control for loading. (B) Growth of conditional strain in solid medium. Serial tenfold dilutions of FB1 (WT) and UMC23 (wee1nar) cultures were spotted in solid rich medium (YPD) and minimal medium with nitrate (MMNO3). YPD plates were incubated for 2 days and the nitrate plates for 4 days at 28°C. (C) Western blotting of inhibitory phosphorylation following wee1 depletion. Protein extracts from the strains indicated were obtained after incubation in repressive conditions (YPD) at the times indicated (in hours). The Cdk1 levels were determined with anti-phospho-Cdc2 (Tyr15) and anti-PSTAIRE antibodies. (D) Flow cytometry of wild-type and UMC23 cells grown in permissive and restrictive conditions. Cells grown in MMNO3 were centrifuged, washed twice in minimal medium without nitrogen, and resuspended in the appropriate medium. Samples were taken for FACS analysis at the times indicated. (E) Length of wild-type and UMC23 cells growing in permissive conditions (MMNO3). In the upper plot, the length of the major axis of FB1 and UMC23 mother cells was measured and plotted as function of the number of cells. Below, the length of the buds was measured from the same population. A sample of 110 cells was used for each measurement.

View larger version (52K): [in a new window]   Fig. 6. Morphology of wee1 conditional cells. (A) Wild-type and mutant cells in permissive and restrictive conditions. FB1 (wild type) and UMC23 (wee1nar) cells were incubated for 8 hours in MMNO3 or YPD and then stained with DAPI and FITC-WGA to visualize their nuclei and cell walls, respectively. Scale bars: 20 µm.

View larger version (60K): [in a new window]   Fig. 7. Relationship between Clb2 and Wee1. (A) Epistatic analysis of clb2 and wee1. The single mutant TAU42 (clb2nar) and UMC23 (wee1nar) strains, and the double mutant strain UMP40 (clb2na r wee1nar) were grown in restrictive conditions (YPD) for 8 hours. Note that the phenotype of the double mutant in these conditions is similar to the phenotype of the clb2nar cells. Scale bars: 20 µm. (B) Overexpression of wee1 overcomes the morphological effects imposed by high levels of Clb2. UMC45 cells that express constitutively high levels of clb2 and carry the ectopic arabinose-inducible Pcrg:wee1 allele, grow as filaments with short cell compartments in non-inducing conditions (YPD). Transferring the same cells to inducing conditions (YPA) produces elongated cells that resemble wild-type cells overexpressing wee1. Scale bars: 20 µm). (C) Inhibitory phosphorylation of B cyclin-associated Cdk1. Protein extracts from cells expressing epitope-tagged versions of Cln1 (SONU58), Clb1 (UMP19) and Clb2 (UMP27) were immunoprecipitated with anti-myc (Cln1 and Clb2 extracts) or anti-VSV (Clb1 extract). Whole cell extracts (WCE) and immunoprecipitates (IP) were immunoblotted with anti-phospho-Cdc2 (Tyr15) and anti-PSTAIRE antibodies. Cdk2 is the U. maydis homolog of S. cerevisiae Pho85, which is also recognized by the anti-PSTAIRE antibody.

View larger version (38K): [in a new window]   Fig. 8. Cells in which wee1 is transcriptionally deregulated, do not adapt to different nutritional conditions. (A) The levels of wee1 mRNA are transcriptionally regulated by nutritional conditions. Wild-type FB1 cells (WT) and the mutant UMC44 strain (wee1scp) in which the endogenous wee1 promoter has been exchanged for the scp promoter, were grown in nutrient-rich (YPD), complete (CMD) and minimal medium (MMNO3) to OD600 0.5. Their RNA was isolated and 10 µ g of RNA was loaded per lane, using 18s rRNA as a loading control. (B) Length of wild-type and wee1scp cells growing in different media. The length of the major axis of 120 cells was measured and plotted as a function of the number of cells. (C) DNA content of wild-type and wee1scp cells growing in different media.

View larger version (56K): [in a new window]   Fig. 9. Inhibitory phosphorylation of Cdk1 is required for infective tube formation. (A) Mixtures of the strains indicated were spotted on MMNH4-charcoal (non-inducing) and MMNO3-charcoal (inducing) plates, and incubated for 48 hours at room temperature. Fuzziness (gray colonies) was an indication of successful infective tube formation. (B) Morphology of the solopathogenic strain SG200 and its derivatives, UMC14 (SG200 Pnar1:cdk1) and UMC13 (SG200Pnar1:cdk1AF), which were incubated in inducing conditions (liquid MMNO3 medium) for 8 hours. Scale bars: 20 µm.

View larger version (123K): [in a new window]   Fig. 10. Impaired inhibitory phosphorylation of Cdk1 affects the ability of U. maydis to produce a successful plant infection. (A) Disease symptoms on the leaf blades of young maize plants 14 days post-inoculation with the strains indicated. Control infection (a1 b1 Pmig1:cdk1 x a2 b2) produced the characteristic symptoms of disease (observe the red stain produced by the anthocyanin streaking and small tumors at the leaf margin). In contrast, infection with cells expressing the cdk1 allele refractory to Tyr15 phosphorylation (a1 b1 Pmig1:cdk1AF x a2 b2) did not induce symptoms other than chlorosis (observe the yellowing of the leaf tissue). (B) Hyphal development of a1 b1 Pmig1:cdk1 x a2 b2 (upper panel) and a1 b1 Pmig1:cdk1AF x a2 b2 (bottom panel) cells inside the plant tissue. Symptomatic leaves were removed one week after inoculation and the presence of fungal cells was detected by staining with Chlorazole Black E. Observe the dramatic difference in growth between the a1 b1 Pmig1:cdk1AF x a2 b2 hyphae (bottom panel) and the controls, where two branching hyphae can clearly be seen (upper panel). Scale bars: 20 µm.