Serial Analysis of Gene Expression Reveals Conserved Links between Protein Kinase A, Ribosome Biogenesis, and Phosphate Metabolism in Ustilago maydis

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.Development and virulence are regulated by cyclic AMP (cAMP)/protein kinase A (PKA) and mitogen-activated protein (MAP) kinase signaling pathways in several fungi, including the corn smut pathogen Ustilago maydis (13,38,46). Mating of haploid U. maydis cells, which are nonpathogenic and yeast-like, leads to the formation of infectious, dikaryotic hyphae. This process is regulated by two unlinked mating-type loci, a and b. Cell recognition and fusion of conjugation tubes are controlled by the a mating-type locus encoding a pheromone (mfa)-pheromone receptor (pra) system that acts through a conserved MAP kinase module (8). Several components of the MAP kinase module have been identified, including the MAP kinase kinase kinase Ubc4, the MAP kinase kinase Fuz7/Ubc5, the MAP kinase Ubc3/Kpp2, and the putative adaptor protein Ubc2 (1,5,(46)(47)(48)50).

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

“…The transport activity is not essential for the sensor function of Pho84, and targets of the PKA pathway are known to be regulated by the sensing activity of Pho84, thus indicating an influence on PKA activity (51,52,86,87). Our previous work also revealed that deletions in genes encoding components of PKA perturbed phosphate metabolism and polyphosphate formation in U maydis (35,36). Pho84 (and particularly Pho840) may also have a sensing function and influence PKA activity in C. neoformans.…”

“…In S. cerevisiae, Vtc4 was previously identified as a key component of the polyphosphate polymerase in the vacuolar transport chaperone complex that synthesizes polyphosphate at the vacuolar membrane (33,34). In this context, we previously showed that cAMP/PKA signaling influenced the expression of genes for phosphate uptake and storage in the fungal pathogen of maize, Ustilago maydis (35). One of the genes encoded Vtc4, and deletion of this gene reduced polyphosphate formation, influenced the transition between yeast and filamentous growth, and attenuated virulence (36).…”

Proteomic analysis of dimorphic transition in the phytopathogenic fungus Ustilago maydis

“…Others have been identified in a screen for genes regulated by cAMP-signalling (e.g. the aldo-keto reductase YakC, actin [20] and SEP3 [21]. However, additional proteins, not yet detected in other screens were also found to respond to b-locus induction.…”

“…Two genes encoding catalytic subunits of protein kinase A (adr1 and uka1) have also been identified as part of cAMP signalling [38,39]. In the U. maydis adr1 mutant strain the septin SEP3 has been identified as being up-regulated in these filamentously growing cells [21]. The cAMP pathway thus suppresses expression of these genes.…”

The vtc4 Gene Influences Polyphosphate Storage, Morphogenesis, and Virulence in the Maize Pathogen Ustilago maydis

“…Serial analysis of gene expression (SAGE) was previously performed to compare the transcriptomes of wild-type cells versus the PKA mutants ubc1 and adr1 (22). Mutation of the ubc1 gene, encoding the regulatory subunit of PKA, results in a multibudded phenotype.…”