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First published online August 17, 2004
doi: 10.1242/10.1242/jcs.01304

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A member of the Fizzy-related family of APC activators is regulated by cAMP and is required at different stages of plant infection by Ustilago maydis

Sonia Castillo-Lluva^*, Tatiana García-Muse^* and José Pérez-Martín

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

View larger version (46K): [in a new window]   Fig. 1. Cru1 belongs to the Fizzy-related family of APC activators. (A) The Cru1 protein. The seven WD repeats are shown, as well as the putative C-box (DRFIPQR). (B) Comparison of the predicted amino acid sequence encoded by the cru1 gene with the amino acid sequences of S. cerevisiae Hct1/Cdh1 and S. pombe Ste9/Srw1 proteins. Gray boxes indicate identity. Notice that homology between these proteins extends beyond the seven WD repeats. (C) Dendrogram of fungal APC activators. U. maydis Cru1 groups in the same branch as other Fizzy-related activators. Bar, 0.05 substitutions per aa.

View larger version (60K): [in a new window]   Fig. 2. Ectopic expression of cru1. (A) Growth ability of cells expressing an ectopic copy of cru1 gene was examined by spotting serial dilutions of exponential cultures of UMP28 and TAU54 strains in solid Complete Medium with either 2% glucose (CMD, noninduction conditions) or 2% arabinose (CMA, induction conditions). Plates were incubated for 3 days at 28°C. (B) Micrographs showing the cell morphology of TAU54 cells after 9 hours of growth in CMA liquid cultures (inducing conditions). Notice the elongated shape, and the presence of a single nucleus (DAPI staining) and a microtubule network compatible with a G2-like state (GFP-Tub1 epifluorescence). Bars, 10 µm. (C) FACS analysis of DNA content of UMP28 and TAU54 cells in noninducing conditions (CMD) and inducing conditions (CMA). Samples were removed after 0, 3, 6 and 9 hours after transfer to conditional medium. The shift to higher than 2C DNA content observed in TAU54 cells incubated in CMA after 9 hours is due to mitochondrial DNA staining. (D) Western analysis of cyclin levels after cru1 overexpression. Protein extracts from the indicated strains, carrying epitope tagged versions of U. maydis B-type cyclins, were obtained after incubation in noninducing (CMD) and inducing (CMA) conditions at the indicated times (in hours). Clb1 was detected by using an anti-VSV antibody, whereas Clb2 was detected with an ani-MYC antibody. As loading control we used Cdk1 levels, detected with an anti-PSTAIRE antibody.

View larger version (52K): [in a new window]   Fig. 3. Phenotypic characterization of cru1 cells. (A) Morphology of cru1 cells. Wild-type (FB1) and cru1 (UMP7) cells growing in YPD medium until exponential phase were observed by phase contrast and DAPI staining. Bars, 10 µm. (B) Length distribution of wild-type (FB1) and cru1 (UMP7) cells growing in CMD medium until exponential phase. In the upper plot, the length of the major axis of 150 FB1 mother cells and 130 UMP7 mother cells was measured, and plotted as a function of the number of cells. In the bottom plot the length of the buds was measured from the same population. In the UMP7 population all cells showed buds, whereas in the FB1 population only 93 cells were budded. (C) DNA content of wild-type and cru1 cells growing in different media. (D) Wild-type and cru1 cells carrying a VSV-tagged version of Clb1 were arrested with benomyl at G2/M transition. After the release in benomyl-free medium, samples were taken at the indicated times and both FACS analysis and protein extracts were obtained. Clb1 was detected with anti-VSV antibody. As loading control Cdk1 levels were used.

View larger version (26K): [in a new window]   Fig. 4. Cru1 is required for adaptation to nitrogen starvation. (A) The cru1 cells cannot arrest in the G1 phase upon nitrogen starvation. Wild-type and mutant strains growing in nutrient-rich medium until mid-exponential phase were transferred to minimal medium without nitrogen (MM-N) for the duration indicated and analyzed by flow cytometry. (B) DAPI staining of wild-type and mutant cells after 8 hours incubation in nitrogen starvation conditions. Notice the presence of a single nucleus per cell. Bars, 10 µm. (C) Survival of wild-type and mutant cells in medium lacking nitrogen after 3 days. Cells were grown in nutrient-rich medium to around 106 cells/ml, washed, resuspended in MM-N and incubated at 28°C. Aliquots of each culture were extracted at the indicated times and cells were counted on YPD plates to determine the number of viable cells.

View larger version (42K): [in a new window]   Fig. 5. The mRNA levels of cru1 are regulated by cAMP/PKA. (A) The levels of cru1 mRNA are regulated by nutritional conditions. FB1 cells were grown in nutrient-rich medium (YPD), complete medium (CMD) and minimal medium (MMD) until OD600 0.5. RNA was isolated and 10 µg of RNA was loaded per lane. The same filters were hybridized in succession with the probes indicated on the left. A quantification of the cru1 signal relative to the rRNA signal is shown on the right. (B) Northern analysis of cru1 mRNA levels in response to cAMP. FB1 cells were grown for 6 hours in CMD containing the indicated concentrations of cAMP, and 10 µg of total RNA was loaded per lane. Quantification of the relative signal is shown on the right. (C) Levels of cru1 mRNA in cAMP/PKA mutants. Total RNA from the strains indicated on the top growing in CMD until mid-exponential phase was isolated and subjected to northern analysis with the probes indicated on the left. Quantification of the relative signal is shown on the right.

View larger version (106K): [in a new window]   Fig. 6. Cru1 is required for full virulence. (A) Tumors produced by wild-type strains after 2 weeks of infection. (B) Tumors produced by cru1 cells after 2 weeks of infection. Further incubation does not result in an increase in tumor size. (C) Section of a 3-week tumor produced by wild-type strains. A massive production of mature teliospores can be observed. The inset shows a higher magnification of teliospores. Bar, 10 µm. (D) Section of a 3-week tumor produced by cru1 cells. Very few mature teliospores can be observed and frequently they have an anomalous morphology (inset showing a higher magnification. Bar, 10 µm).

View larger version (98K): [in a new window]   Fig. 7. Cells defective in cru1 fail to grow inside the plant. (A) Disease symptoms on the leaf blades of young maize plants 14 days post inoculation with the strains are indicated. Wild-type infection produced the characteristic symptoms of disease (antocyanin streaking and tumor production); however, cru1 cell infections do not induce symptoms further to chlorosis. Notice the chlorotic area near the infection point (arrow). (B) Clorazole Black E staining of symptomatic leafs after 1 week of inoculation. Notice the proliferation of wild-type cells and the absence of such growth in mutant cells. Bars, 15 µm.

View larger version (78K): [in a new window]   Fig. 8. Cru1 is required for the expression of the mfa1 gene. (A) Mixtures of the indicated strains were spotted on PD-charcoal plates and incubated for 48 hours at room temperature. Fuzziness was an indication of a successful mating. (B) Formation of conjugative tubes by autocrine cells. Cultures of the autocrine strain TAU3 growing in CMD at OD600 of 0.2 were treated with 0.1 ng/ml of synthetic a1-mating pheromone for 8 hours, resulting in the induction of conjugation tubes. A similar treatment of the TAU3 cru1 derivate (TAU7) resulted in no response. Treatment of TAU3 cru1 with 1 µg/ml of synthetic pheromone induces the formation of hyphal extensions that resemble conjugation tubes. Bars, 10 µm. (C) Absence of mfa1 expression in Cru1-defective cells. Cultures of TAU3 and TAU7 cells were treated with 0.1 ng/ml of synthetic a1-mating pheromone and samples were obtained at the indicated times (in minutes). RNA was isolated and 10 µg of RNA was loaded per lane. The blot was probed with mfa1 and rRNA.

View larger version (66K): [in a new window]   Fig. 9. The expression of the mfa1 gene is regulated by the levels of Cru1 and Clb1. (A) Overexpression of cru1 induces mfa1 expression in nutrient-rich medium. The strains indicated on the top were incubated for 4 hours in YPD (noninducing conditions) or YPA (inducing conditions). RNA was isolated and 10 µg of RNA was loaded per lane. The blot was probed with mfa1, cru1 and rRNA. The cru1 mRNA appears as two different populations indicated by arrows: Pcru1 is the mRNA expressed from the native cru1 promoter and Pcrg1 is the mRNA expressed from the chimeric construction inserted in the ip locus. The different size is the result of the different transcription start point in both promoters. (B) Depletion of Clb1 bypassed the requirement of Cru1. The strains indicated on the top were grown in minimal medium with NO3 (permissive conditions for the clb1nar allele) until OD600 of 0.2 and then were incubated for 4 hours in CMD (restrictive conditions for the clb1nar allele). RNA was isolated and 10 µg of RNA was loaded per lane. The blot was probed with mfa1 and rRNA.

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