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First published online 16 December 2003
doi: 10.1242/jcs.00877

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Characterization of B-type cyclins in the smut fungus Ustilago maydis: roles in morphogenesis and pathogenicity

¹ Department of Microbial Biotechnology, Centro Nacional de Biotecnología CSIC, Campus de Cantoblanco-UAM, 28049 Madrid, Spain
² Max-Planck-Institut für Terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany

View larger version (63K): [in a new window]   Fig. 1. Characterization of Cdk1, the catalytic subunit of the mitotic kinase from Ustilago maydis. (A) Scheme of the Cdk1 protein. Cdk1 contains all motifs typical for members of the Cdc2 family. This includes Tyr15, which is the putative Wee1 phosphorylation site; a PSTAIRE domain; and Thr167, which is the putative CAK phosphorylation site. (B) Alignment of selected members of the fungal Cdc2 family. Accession numbers for these proteins are: S. pombe Cdc2: AAA35293; U. maydis Cdk1: AY260971; S. cerevisiae Cdc28: NP009718, Candida albicans Cdc28: P43063. (C) Identification of the Cdk1 molecule. Lysates were prepared from exponential cultures of wild-type FB1 (cdk1), and tagged-strain TAU17 (cdk1-1) cells. The whole cell lysates were separated by SDS-PAGE and immunoblotted with anti-PSTAIRE (upper panel) or anti-FLAG (lower panel) antibodies. (D) The Cdk1 protein binds Suc1. The same lysates used in C were incubated with Suc1-beads to pull down Cdc2-like proteins. Precipitates were immunoblotted with anti-PSTAIRE and anti-FLAG antibodies. (E) Immunoprecipitation of Cdk1 tagged with the FLAG epitope. Lysates from FB1 (cdk1) and TAU17 (cdk1-1) cells were subjected to immunoprecipitation with the anti-FLAG M2 antibody. Crude extract or immunoprecipitates (FLAG IP) were immunoblotted with anti-PSTAIRE. (F) H1-histone kinase activity of the immunoprecipitates recovered in E.

View larger version (37K): [in a new window]   Fig. 2. Characterization of B-type cyclins from U. maydis. (A) Scheme of Clb1 and Clb2 proteins. U. maydis cyclins contained the FLRRXSK motif (white boxes) and `destruction box' consensus sequences (gray boxes). (B) Dendrogram of selected B-type cyclins from fungi. U. maydis Clb1 groups with the S- and M-phase cyclins from S. pombe, while U. maydis Clb2 groups with a heterogeneous B-type cyclins. Accession numbers for the proteins are: ScClb5, P30283; CaCyb1, U40430; ScClb2, S14166; SpCig1, P24865; CaCyb4, AAC79857; ScClb3, A60048; UmClb2, AY260970; UmClb1, AY260969; SpCig2, P36630; SpCdc13, P10815. Bar: 0,05 substitutions per aa. (C) Clb1 and Clb2 are highly abundant in S- and G2/M-arrested cells, but present at low levels in G1 cells. Extracts from cells carrying an epitope-tagged copy of Clb1 and Clb2 were analyzed by immunoblotting. Samples are as follows: G1, culture enriched in G1 phase cells; Ben, cells arrested at G2/M transition after treatment with benomyl for 1 hour; HU, cells arrested in S phase by treatment with hydroxyurea for 90 minutes; As, asynchronous cells. The same filters were also probed with anti-PSTAIRE antibodies (bottom panel), showing that the abundance of Cdk1 varied less than twofold. (D) Clb1 associates with Cdk1. Lysates prepared from wild-type FB1 and Clb1-tagged UMP19 (clb1-1) cells were incubated with Suc1 beads to pull down Cdk1 (upper panel) or were immunoprecipitated with anti-VSV antibody to pull down Clb1 (lower panel). The whole cell lysates as well as the precipitates were separated by SDS-PAGE and immunoblotted with anti-PSTAIRE and anti-VSV to detect Cdk1 and Clb1 proteins respectively. (E) Clb2 associates with Cdk1. Lysates prepared from wild-type FB1 and Clb2-tagged UMP27 (clb2-1) cells were incubated with Suc1 beads to pull down Cdk1 (upper panel) or were immunoprecipitated with anti-MYC antibody to pull down Clb2 (lower 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 Clb2 proteins respectively. The different bands with higher electrophoretic mobility detected in the anti-MYC IP are degradation products of Clb2.

View larger version (56K): [in a new window]   Fig. 3. Conditional removal of cyclins. (A) Levels of clb1 and clb2 mRNA in the conditional strains. Wild-type FB1 and conditional strains TAU41 (FB1 clb1nar) and TAU42 (FB1 clb2nar) were grown for 3 hours in permissive conditions [minimal medium with nitrate (NO3)] or restrictive conditions [minimal medium with ammonium (NH4), or rich medium, (YPD)] and then RNA was extracted and subjected to northern analysis, after loading 10 µg total RNA per lane. The filters were hybridized with probes for clb1 or clb2. A probe for 18s rRNA was used as loading control. (B) Growth of conditional strain in solid medium. Serial tenfold dilutions of FB1, TAU41 (FB1 clb1nar) and TAU42 (FB1 clb2nar) cultures were spotted on solid rich medium (YPD), and minimal medium amended with nitrate (NO3) or ammonium (NH4). YPD and ammonium plates were incubated for 2 days, while nitrate plates were incubated for 3 days. All incubations were at 28°C. (C) Flow cytometry analysis of wild-type, TAU41 and TAU42 cells grown in permissive and restrictive conditions. Cells grown in MM-NO3 were centrifuged, washed twice in minimal medium without nitrogen, and resuspended in the appropriated medium. Samples were taken for FACS analysis at the indicated times. Because of the shorter generation time of cells in rich medium, samples were taken every hour for a total of 3 hours.

View larger version (61K): [in a new window]   Fig. 4. Morphology of clb1 conditional cells. (A) Morphology of the wild-type and mutant cells in restrictive conditions. FB1 and UMP25 cells were incubated for 9 hours in MM-NH4. Two clear morphologies can be seen: unbudded and budded cells, all of them with a single nucleus per cell (right panel shows DNA stained with DAPI). Scale bar: 10 µm. (B) Microtubule organization in UMP25 cell arrested after 6 hours in rich medium. Note that in arrested cells long microtubules reach from the neck to the growing tip. (C) UMP25 cells incubated for 25 hours in rich medium have a distinct phenotype characterized by a extensive polarized growth (upper horizontal panel) disorganized tubulin cytoskeleton (middle horizontal panel) and empty sections behind that are separated by septa (stained with calcofluor in lower horizontal panel, arrows point the septa) generated by formation of basal vacuoles (arrow; vertical panel). Scale bars: (horizontal panels) 10 µm; (vertical panel) 5 µm.

View larger version (60K): [in a new window]   Fig. 5. Morphology of clb2 conditional cells. (A) Quantification of FB1 (WT) and UMP26 (clb2nar) cells growing in different media showing the proportion of unbudded cells, cells with a bud shorter than the mother cell and cells with a bud larger than the mother cell. More than 100 cells were recorded in each condition. (B) UMP26 cells were grown in rich medium for 6-9 hours and cell morphology (B1), microtubules organization (B2), and nuclear morphology (C, DAPI staining) were observed. Scale bar: 10 µm.

View larger version (54K): [in a new window]   Fig. 6. Consequences of Clb2 overexpression. (A) Cells overexpressing a wild-type copy of Clb2 under the control of the Pcrg1 promoter (TAU52-1 cells) were grown in CM with 2% arabinose for 9 hours and samples were DAPI stained (upper panel), or WGA stained to detect the presence of septa (lower panel). Scale bars: 10 µm. (B) Flow cytometry analysis of wild-type and TAU52-1 (FB1 Pcrg-clb2) cells grown in no induction (CMD) and induction (CMA) conditions. Cells grown in CMD were centrifuged, washed twice in water, and resuspended in the appropriated medium. Samples were taken for FACS analysis at the indicated times. The extra peak in TAU52 cells grown in CMA corresponds to a 4C DNA content. (C) Cell morphology of TAU26 (FB1 Phsp70-clb2) cells growing in liquid CMD medium after 24 hours of incubation. Right panel show DAPI staining. Scale bar: 10 µm.

View larger version (50K): [in a new window]   Fig. 7. Consequences of Clb1 overexpression. (A) Cell morphology of TAU36-1 cells growing in YPA (inducing conditions). Observe in the cells stained with DAPI the diverse intensity of fluorescence from various nuclei. Scale bar: 10 µm. (B) Flow cytometry analysis of wild-type (FB1) and TAU36-1 cells grown in no-induction (YPD) and induction (YPA) conditions. Cells grown in YPD were centrifuged, washed twice in water and resuspended in the appropriated medium. Samples were taken for FACS analysis at the indicated times. Note the peaks with DNA content below 1C and above 2C. (C) Serial tenfold dilutions of exponential cultures in YPD of wild-type FB1 and TAU36-1 (FB1 Pcrg-clb1) cells were spotted on induction plates (YPA, YP medium with 2% arabinose) with 5 µg/ml phloxin B and incubated at 28°C for 4 days. Phloxin B accumulates in dead cells and subsequently red colonies are indicative of a loss of viability. (D) Benomyl sensitivity of strain overexpressing Clb1. Serial tenfold dilutions of exponential cultures of FB1 and TAU36-1 cells were spotted on induction plates containing 0.6 µg/ml of benomyl. Plates were incubated at 28°C for 4 days.

View larger version (52K): [in a new window]   Fig. 8. Clb1 and Clb2 proteins carry functional destruction boxes. (A) Scheme showing the Clb1 and Clb2 proteins, as well the different deletion mutants. The sequences for the destruction boxes are indicated. These constructs were expressed under the control of a less-active version of the Pcrg1 promoter to avoid the problems associated to high cyclin levels (see text for details). (B) Expression levels of Clb1 and Clb2 derivates. Cells were grown in non-induction (complete medium with 2% glucose, CMD) and induction (complete medium with 2% arabinose, CMA) conditions for 6 hours. Whole cell lysates were separated by SDS-PAGE and immunoblotted with anti-VSV (Clb1 western, upper panel) or anti-MYC (Clb2 western, lower panel) antibodies. Similar amount of total extract was loaded per lane. Upper panel: TAU36-2 (Clb1), TAU30 (Clb1db1), TAU45 (Clb1db2) and TAU31 (Clb1db1-2) cells that express VSV-tagged versions of Clb1 and derived proteins. The different level of protein expression could be attributed to a lower translation efficiency in the derivates lacking the N-terminal destruction box. Lower panel: TAU52-2 (Clb2) and TAU53 (Clb2db) cells that express MYC-tagged versions of Clb2 and Clb2db proteins. (C) Serial tenfold dilutions of exponential cultures of FB1 (control), TAU36-2 (Pcrg*-Clb1), TAU30 (Pcrg*-Clb1db1), TAU45 (Pcrg*-Clb1db2), TAU31 (Pcrg*-Clb1db1-2), TAU52-2 (Pcrg*-Clb2), and TAU53 (Pcrg*-Clb2db) strains were spotted on complete medium plates with glucose (CMD) or arabinose (CMA) as sole carbon source. Plates were incubated at 28°C for 3 days. (D) Deletion of D-boxes stabilizes Clb1 and Clb2. TAU36-2, TAU31, TAU52-2 and TAU53 cells were grown for 1 hour in complete medium with 2% arabinose, and then transferred to completed medium with 2% glucose and 100 µg/ml cycloheximide. Samples were taken at the indicated time, and Clb1, Clb2 and Cdk1 levels were analysed by immunoblotting with anti-VSV, anti-MYC and anti-PSTAIRE antibodies respectively.

View larger version (63K): [in a new window]   Fig. 9. Effects of the expression of a stable version of Clb1. (A) Cell morphology of TAU57 strain grown in CMA for 9 hours. This strain carries the clb1db1-2 transgene and a Cfp-tub1 fusion. All cells were arrested with a bud about the size of the mother cell. Three different condensed nucleus morphologies were apparent: a single nucleus near the neck, two nuclei nearby and a nucleus split in two located in the neck (arrows point to nuclei). Scale bar: 10 µm. (B) Cells expressing Clb1db1-2 arrested with a 2C DNA content. Flow cytometry analysis of wild-type FB1 and TAU57 cells grown in no-induction (CMD) and induction (CMA) conditions. Cells grown in CMD were centrifuged, washed twice in water, and resuspended in the appropriated medium. Samples were taken for FACS analysis at the indicated times. (C) Microtubule organization in TAU57 cells growing in non-induction conditions (CMD) and induction conditions (CMA). Observe the absence of microtubule structures in inducing conditions.

View larger version (65K): [in a new window]   Fig. 10. Effects of the expression of a stable version of Clb2. (A) Cells expressing Clb2db arrested with a 2C DNA content. Flow cytometry analysis of wild-type and TAU58 cells grown in no-induction (CMD) and induction (CMA) conditions. Cells grown in CMD were centrifuged, washed twice in water, and resuspended in the appropriated medium. Samples were taken for FACS analysis at the indicated times. (B) TAU58 cells grown in CMA for 9 hours were stained with DAPI to observe nuclei. (C) TAU58 cells grown for 9 hours in CMD or CMA were stained with WGA to detect the presence of septum in the cells. Note that no septa are apparent in cells growing in CMA. (D) Formation of the mitotic spindle in TAU58 cells treated as before, observed with epifluorescence. Compare the MT pattern in non-induction conditions (CMD) and induction conditions (CMA). Scale bars: 5 µm.

View larger version (102K): [in a new window]   Fig. 11. Influence of high levels of Clb2 in mating and pathogenicity. (A) Disease symptoms on the leaf blades of young maize plants 14 days post inoculation with the strains indicated. Observe the anthocyanin production (arrow a) and small tumors (arrow t) in the wild-type infection and the chlorosis (yellowing of the green tissue of the leaf, arrow c) in the mutant infection. (B) Mating assay of Clb2-overexpressing cells and wild-type strains. The appearance of white filaments indicates formation of dikaryotic hyphae. Note that the mutant cross produced fewer hyphae. (C) Dikaryotic hypha resulting from a wild-type FB1 x FB2 cross. Note the empty compartments to the right of the elongated tip cell. (D) Dikaryotic hypha resulting from a mutant TAU26 x FB2 cross. Note the `spiky' appearance of the elongated tip cell. (E) Hyphal development of wild-type cells inside the plant tissue. Cross walls separate the long cylindrical cells of hyphe. The inset shows a lower magnification of the filamentous network. (F) Hyphal development of mutant cells inside the plant tissue. These cells appeared to generate incipient branches at a higher frequency than wild-type cells. Scale bars: 50 µm (B,C); 20 µm (E-F).

View larger version (111K): [in a new window]   Fig. 12. Infectivity of diploids carrying a single dose of B-cyclin genes. (A) Morphology of a wild-type diploid cell (inset, FBD11 cells at same magnification) and a diploid cell with a single gene dose of clb2 (UMP32). Cells were grown in CMD to exponential phase and DAPI stained. Scale bar: 10 µm. (B) Charcoal assay of diploid strains heterozygous for mating type. FBD11 is a wild-type strain, UMP21 has a single dose of the clb1 gene, and UMP32 has a single dose of the clb2 gene. (C) Disease symptoms on the leaf blades of young maize plants 14 days post inoculation with the strains indicated. Strong anthocyanin production was induced by infection with UMP32 but no tumors were apparent. (D) Hyphal development of wild-type, UMP21 and UMP32 cells inside the plant tissue. Observe that mutant UMP32 cells appeared to have fewer branches and the hyphae look wider the wild-type hyphae. Scale bars: 20 µm.