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Comparative Study
. 2005 Dec;4(12):2029-43.
doi: 10.1128/EC.4.12.2029-2043.2005.

Serial analysis of gene expression reveals conserved links between protein kinase A, ribosome biogenesis, and phosphate metabolism in Ustilago maydis

Luis M Larraya  1 , Kylie J Boyce, Austin So, Barbara R Steen, Steven Jones, Marco Marra, James W Kronstad
Affiliations
Comparative Study

Serial analysis of gene expression reveals conserved links between protein kinase A, ribosome biogenesis, and phosphate metabolism in Ustilago maydis

Luis M Larraya et al. Eukaryot Cell. 2005 Dec.

Abstract

The switch from budding to filamentous growth is a key aspect of invasive growth and virulence for the fungal phytopathogen Ustilago maydis. The cyclic AMP (cAMP) signaling pathway regulates dimorphism in U. maydis, as demonstrated by the phenotypes of mutants with defects in protein kinase A (PKA). Specifically, a mutant lacking the regulatory subunit of PKA encoded by the ubc1 gene displays a multiple-budded phenotype and fails to incite disease symptoms, although proliferation does occur in the plant host. A mutant with a defect in a catalytic subunit of PKA, encoded by adr1, has a constitutively filamentous phenotype and is nonpathogenic. We employed serial analysis of gene expression to examine the transcriptomes of a wild-type strain and the ubc1 and adr1 mutants to further define the role of PKA in U. maydis. The mutants displayed changes in the transcript levels for genes encoding ribosomal proteins, genes regulated by the b mating-type proteins, and genes for metabolic functions. Importantly, the ubc1 mutant displayed elevated transcript levels for genes involved in phosphate acquisition and storage, thus revealing a connection between cAMP and phosphate metabolism. Further experimentation indicated a phosphate storage defect and elevated acid phosphatase activity for the ubc1 mutant. Elevated phosphate levels in culture media also enhanced the filamentous growth of wild-type cells in response to lipids, a finding consistent with PKA regulation of morphogenesis in U. maydis. Overall, these findings extend our understanding of cAMP signaling in U. maydis and reveal a link between phosphate metabolism and morphogenesis.

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Figures

FIG. 1.
FIG. 1.
Evaluation of RNA for SAGE library preparation. The RNA quality for the wild-type strain 002 (lane WT) and the mutant strains 111 (ubc1::hygBr) and 002-10 (adr1::phelor) was tested prior to library construction. (A) rRNA (18S and 28S) bands as a loading control. (B) Hybridization with the constitutively expressed gene for succinate dehydrogenase (cbx). (C) Hybridization with the pheromone gene (mfa1).
FIG. 2.
FIG. 2.
Sensitivity of the adr1 mutant to rapamycin. Evaluation of the growth of wild-type U. maydis strain 521 and the mutants 111 (ubc1::hygBr) and 002-10 (adr1::phelor), after 2 days at 30°C on PDA plates (A) and PDA plates containing 200 ng/ml rapamycin (B). The growth of strain 002-10 is dramatically reduced on plates containing rapamycin.
FIG.3.
FIG.3.
Influence of phosphate on the morphology of U. maydis in Tween 40. Differential-interference-contrast micrographs of the wild-type U. maydis strain 002 (a1b1) grown in minimal medium (minus phosphate) supplemented with 1% glucose, 1% glucose plus 1% Tween 40, or 1% Tween 40. Cultures were also supplemented with 1, 7.35, or 250 mM KH2PO4. (A) Budding growth for the wild-type U. maydis strain 521 after 3 days in 1% glucose and 1% glucose plus 1% Tween 40 under all phosphate concentrations tested. In 1% Tween 40, increasing phosphate concentration results in increased filamentation. U. maydis grows as budding yeast cells at low phosphate concentrations (1 mM), begins to extend cell length in 7.35 mM phosphate, and grows as hyphal filaments in high phosphate concentration (250 mM). (B) Budding growth after 5 days in 1% glucose and 1% glucose plus 1% Tween 40 under all phosphate concentrations tested. At this time, however, cells growing in 1% Tween 40 supplemented with 1 mM phosphate remain as budding yeast cells, but the cells are completely filamentous when the medium contains 7.35 mM and 250 mM phosphate. Scale bars are 20 μm.
FIG. 4.
FIG. 4.
Reduced polyphosphate accumulation in the ubc1 mutant. Differential-interference-contrast micrographs of the wild-type strain 521 and the ubc1 and adr1 mutants grown overnight in PDB and stained for polyphosphate with toluidine blue O dye are shown. The ubc1 mutant showed a decrease in the amount of polyphosphate accumulation compared to that in the wild-type strain and the adr1 mutant. Specifically, 100 cells of each strain from separate experiments were scored as stained or unstained with the following results (percentages of stained cells): strain 521, 99% ± 0.0%; adr1 mutant, 98% ± 2.8%; and ubc1 mutant, 74% ± 6.4%. Scale bars = 20 μm.
FIG. 5.
FIG. 5.
RNA blot analysis of selected genes with differentially expressed tags among the WT, UBC1, and ADR1 libraries. (A) rRNA (18S and 28S) bands as a loading control. (B) Hybridization patterns of representative genes for which tags were elevated in the UBC1 library and/or down-regulated in the ADR1 library. (C) Hybridization of representative genes with tags with lower levels in the UBC1 library and/or higher levels in the ADR1 library. (D and E) Hybridization of representative genes for which tags were reduced and elevated, respectively, in the libraries from the mutants. The normalized frequencies of the tags for the genes in the three libraries are indicated after the MIPS U. maydis gene accession number in the order WT:UBC1:ADR1.
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