Pheromone‐regulated target genes respond differentially to MAPK phosphorylation of transcription factor Prf1

Abstract

SummaryPheromone signalling during mating is essential for pathogenicity of Ustilago maydis. The activity of the key transcription factor Prf1 is controlled at the transcriptional level and post-translationally by mitogenactivated protein kinase (MAPK) and protein kinase A (PKA) phosphorylation. However, the precise contribution of these regulatory mechanisms to the transcriptional output is unknown. Here, we genetically dissected the three levels of Prf1 regulation. We performed transcriptional profiling of respective mutants to identify and classify targets. This approach revealed that transcriptional regulation of prf1 had only minor influence on target gene expression stressing the importance of post-translational control. PKA regulation of Prf1 was sufficient to control expression of nine pheromone-responsive genes including the major transcription factor regulating pathogenicity. MAPK regulation was necessary for the pheromone response of a set of 57 genes. In 35 cases, pheromone responsiveness was completely lost, while in the remaining 22 cases regulation was alleviated. This indicated a novel level of complexity in MAPK signalling suggesting that target genes respond differentially to MAPK phosphorylation of the respective transcription factors.

Evolution of Mating Systems in Basidiomycetes and the Genetic Architecture Underlying Mating-Type Determination in the YeastLeucosporidium scottii

“…This raises the hypothesis that these genes were lost during evolution as a consequence of gross genomic rearrangements at the P/R locus. Of the three genes, only STE12 is expected to have a mating-related role in L. scottii since homologs of this gene were shown to function as master regulators of the pheromone/receptor-signaling pathway in other fungal species (Sprague and Thorner 1992;Zarnack et al 2008;Jones and Bennett 2011). In C. neoformans, C. amylolentus, and K. mangrovensis, STE12 alleles are matingtype-specific (Findley et al 2009;Guerreiro et al 2013) and, in the former species, this gene is involved in morphogenesis, virulence, and ecological fitness (Chang et al 2001).…”

TheUstilago maydisClp1 Protein Orchestrates Pheromone andb-Dependent Signaling Pathways to Coordinate the Cell Cycle and Pathogenic Development

“…However, the introduction of the mfa2 gene to activate the pheromone pathway (resulting in strain SG200) increased virulence significantly, demonstrating that the interplay between the a and b pathway is required for full virulence (Di Stasio et al, 2009). As outlined, the activation of the a pathway leads to induction of bE, bW, and rbf1 (Urban et al, 1996;Zarnack et al, 2008), the key regulators for pathogenic development, and thus primes the cells for the biotrophic phase. However, there must be a principal difference between the a-dependent rbf1 induction and the subsequent b-dependent rbf1 induction.…”

Tandem KH domains of Khd4 recognize AUACCC and are essential for regulation of morphology as well as pathogenicity inUstilago maydis

“…In order to address this experimentally we investigated whether mRNAs were differentially expressed in the absence of Khd4. Using Affymetrix microarrays (Scherer et al 2006;Zarnack et al 2008; see Materials and Methods), we compared the expression profiles of FB2khd4D and wildtype strain FB2 in two independent transformants and two replicates growing in minimal medium, respectively (Supplemental Table S2). Seventy-two mRNAs were differentially expressed (1.5% of 4786 detected transcripts), whose abundance was significantly changed at least twofold in the deletion strain.…”

“…RNA isolation, sample preparation, and hybridization were performed as previously described (Scherer et al 2006;Zarnack et al 2008). Custom-designed Affymetrix chips based on the U. maydis genome sequence were used Scherer et al 2006).…”

“…Strains were grown to an OD 600 of 0.3 at 28°C in liquid minimal array medium containing glucose and glutamine (Scherer et al 2006;Zarnack et al 2008). RNA isolation, sample preparation, and hybridization were performed as previously described (Scherer et al 2006;Zarnack et al 2008).…”