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First published online 3 April 2007
doi: 10.1242/jcs.005314

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Sustained cell polarity and virulence in the phytopathogenic fungus Ustilago maydis depends on an essential cyclin-dependent kinase from the Cdk5/Pho85 family

¹ Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología CSIC, 28049 Madrid, Spain
² Max-Planck-Institut für Terrestrische Mikrobiologie, Marburg, Germany

View larger version (31K): [in this window] [in a new window]   Fig. 1. Cdk5 in U. maydis. (A) Schematic representation of the U. maydis Cdk5 protein in relation to human Cdk5 (HsCdk5) and S. cerevisiae Pho85 (ScPho85). The catalytic kinase domains are shown as gray boxes and were identified using the Simple Modular Architecture Research Tool (http://smart.embl-heidelberg.de). (B) Dendrogram of CDK-like proteins. The tree was reconstructed using the ClustalW method (http://www.ebi.ac.uk/clustalw/). Bar, 0.02 substitutions per amino acid. Note that U. maydis Cdk5 falls into the Cdk5/Pho85 subfamily of CDKs. An, Aspergillus nidulans; Sp, Schizosaccharomyces pombe; Ca, Candida albicans; Sc, Saccharomyces cerevisiae; Um, Ustilago maydis; Hs, Homo sapiens. (C) Protein levels at different stages of the cell cycle. Extracts from FB1 arrested at S or M phase, G1 phase enriched or an asynchronous culture (As) were assayed by western blot analysis using anti-PSTAIRE antibodies. Both Cdk1 and Cdk5 kinases were detected. (D) Cdk5-GFP appears in the cytoplasm and nucleus. Cells carrying a C-terminal Cdk5-GFP fusion were grown at exponential phase in CMD medium. Subcellular distribution of Cdk5-GFP (upper panels): cells at different phases of the cell cycle showed GFP signal in the nucleus. Only cells carrying large buds (i.e. undergoing mitosis) do not show nuclear fluorescence, probably because of disassembly of the nuclear envelope. In the lower row, DAPI staining co-localizes with the GFP signal. Bar, 10 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 2. Characterization of a temperature-sensitive cdk5ts mutant. (A) Growth of FB1 (wt) and cdk5ts strains at 22°C and 34°C in solid complete medium (CMD). At low temperature the growth of control cells and cdk5ts is similar. However, cdk5ts cells show severe growth defects at 34°C. (B) Morphology of cdk5ts cells after incubation at the restrictive temperature (34°C) for 8 hours. FITC-labeled wheat germ agglutinin (WGA) signal, which marks chitin-rich areas, is restricted to the growing tips in wild-type cells at any temperature, whereas in the cdk5ts mutant the polar staining is lost at high temperature and WGA staining is concentrated in lateral walls. Nuclei were stained with DAPI. Bars, 10 µm. (C) After 12 hours at the restrictive temperature, cells carrying the cdk5ts allele frequently lyse as evidenced by the presence of cell `ghosts' (arrows). By contrast, wild-type cells growing in the same conditions are cigar shaped and form polar buds (inset). Bars, 10 µm. (D) Sensitivity of cdk5ts cells to cell wall inhibitors. Cultures of wild-type (FB1) and cdk5ts strains were grown overnight at the permissive temperature, then diluted to an OD600 of 1.0. Tenfold serial dilutions were made and 2 µl of each was plated in solid YPD medium treated with the indicated compounds. The plates were grown for 4 days at 22°C.

View larger version (67K): [in this window] [in a new window]   Fig. 3. cdk5ts cells are affected in polarity. (A) Calcofluor staining of cdk5ts cells grown in liquid CMD medium for different times at the restrictive temperature (34°C). Note that upon temperature shift, cdk5ts cells expand their diameter and lose their bud constriction, giving cells separated by septation. Bar, 20 µm. (B) Defects in polar growth during G2 arrested cdk5ts cells. Cells overexpressing wee1 arrest at G2 producing elongated buds. However, in cdk5ts cells there is no bud formation and instead two areas of isotropic growth at the poles are apparent. Inset, DAPI staining showing that in cdk5ts cells there is only one nucleus in agreement with a G2 cell cycle arrest. Bars, 10 µm. (C) Effect of cytoskeletal inhibitors on plate growth of wild-type and cdk5ts strains. No difference between cdk5ts and wild-type cells was found in the presence of the MT-destabilizing drug benomyl (1 µM). By contrast, cdk5ts cells are more sensitive to the actin inhibitor cytochalasin D (CytoD; 10 µM).

View larger version (115K): [in this window] [in a new window]   Fig. 4. Actin organization in cdk5ts cells. (A) U. maydis contains a fimbrin-like gene, which is predicted to encode a protein of 615 amino acids that shares a similar domain structure with S. pombe Fim1 (SpFim1) and S. cerevisiae Sac6 (ScSac6). The P-values of the predicted Calponin-homology domains are given in the open boxes. (B) A Fim1-GFP fusion protein localizes to patches within the growing bud of many cells. This localization disappears after treatment with Latrunculin A, indicating that the patches are composed of F-actin. Bar, 3 µm. (C) Fim1-GFP containing actin patches are highly dynamic and move over short distances at the growth region (arrow). Elapsed time is given in seconds. Bar, 2 µm. (D) In temperature-sensitive cdk5ts mutants at the permissive temperature (22°C) Fim1-GFP locates in the bud. Bar, 3 µm. (E) After 4-5 hours at the restrictive temperature (34°C) patches appear at the mother cell and the bud. Bar, 5 µm. (F) Statistics of E. In budding cdk5ts mutant cells at the permissive temperature, most patches are located in the bud (22°C, gray bars), whereas are almost equally distributed between the mother cell and the bud at the restrictive temperature (34°C, black bars).

View larger version (125K): [in this window] [in a new window]   Fig. 5. Distribution of GFP-tagged myosin 5 in cdk5ts mutants. (A) In the control strain FB1, a biologically active GFPMyo5 fusion protein localizes to the growing tip of the bud and the distal cell pole, in which it is thought to prepare the next bud site. Bar, 3 µm. (B) In budding cdk5ts mutant cells at the permissive temperature, GFP-Myo5 is also concentrated at the growing cell poles (arrows). In addition, GFP-Myo5 is found at septa that are, in contrast to control cells, often found in the middle of the mother cell. The polar localization of GFP-Myo5 remains constant over time (compare T=0s and T=16s). Bar, 3 µm. (C) At restrictive temperature (34°C, 4-5 hours), some cdk5ts cells still contain a polar cluster of GFP-Myo5 (arrow at T=0s and box). However, cells only transiently accumulate GFP-Myo5 at the cell pole (arrow in T=16s). In cases where the GFP-Myo5 spot remains at one cell pole, it rapidly rearranges at the putative growth region (arrowheads in detail). Bar, 3 µm. (D) Time series of cell poles of cdk5ts cells show that the GFP-Myo5 accumulation at the growth region remains stationary at the permissive temperature (left series, 22°C), but undergoes rapid rearrangement at restrictive temperature (arrow in right series, 34°C). Elapsed time is given in seconds:milliseconds. Bar, 2 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 6. Cdc24 overexpression bypassed the requirement of Cdk5 for polar growth. (A) Overexpression of cdc24 induces polar growth in cdk5ts cells. Wild-type and cdk5ts cells, as well as derivates carrying constructions for overexpression under the crg1 promoter (repressed in glucose, induced in arabinose) of rac1, rac1Q61L and cdc24 were grown at the permissive temperature until OD600=0.2 and then shifted to the restrictive temperature (34°C) in arabinose-containing complete medium for 5-6 hours. (B) Time series of cell poles of a cdk5ts myo5-GFP cell as well as a cdk5ts myo5-GFP cell overexpressing cdc24 at the restrictive temperature. Note that in the cdk5ts cell the GFP-Myo5 accumulation at the growth region undergoes rapid rearrangement at the restrictive temperature, but when overexpressing cdc24, the GFP-Myo5 signal remains stationary. Elapsed time is given in minutes:seconds. Bars, 3 µm. (C) Lack of suppression of temperature-sensitive growth of cdk5ts cells after cdc24 overexpression. Wild-type and cdk5ts cells carrying a construction overexpressing cdc24 under the control of the crg1 promoter were spotted in inducing (YPA) and non-inducing (YPD) conditions and incubated at permissive (22°C) and restrictive (34°C) conditions.

View larger version (51K): [in this window] [in a new window]   Fig. 7. Cdk5 is required for polar localization of Cdc24. (A) Localization of Cdc24-GFP was analyzed in wild-type and cdk5ts cells grown in CMD at the permissive and restrictive temperatures. In wild-type and cdk5ts cells at the permissive temperature, Cdc24-GFP accumulates at the cell pole. By contrast, in cdk5ts cells at the restrictive temperature, Cdc24-GFP appears to be absent from the bud tip, although a diffuse fluorescence signal was present in the cytoplasm. (B) Quantification of Cdc24-GFP localization at the tip (mean ± s.e.m.; n=80 cells).

View larger version (53K): [in this window] [in a new window]   Fig. 8. Cdk5 is required for virulence. (A) Maize plants were infected with wild-type control strains and cdk5ts mutants and incubated at 22°C and 30°C. The graph shows the quantification (mean ± s.e.m.) of tumor formation on infected maize plants at 22°C and 30°C after 14 days. (B) Mating assays of control strains FB1 x FB2, and cdk5ts mutant strains FB1 cdk5ts x FB2 cdk5ts on charcoal-containing agar plates at 22°C and 30°C. (C) Formation of conjugation hyphae after Fuz7DD overexpression in cells carrying a wild-type cdk5 allele (Pcrg:fuz7DD) and a cdk5ts allele at 30°C. The image on the right shows a cdk5ts cell doublet separated by a septum (arrow). Bar, 20 µm. (D) Formation of infective hyphae after bE/bW overexpression in cells carrying a wild-type cdk5 allele (AB31) and cdk5ts allele at 30°C. Bars, 20 µm.