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A powerful approach to explore gene function is the use of tetracycline-regulated expression. Here, we report the establishment of this titratable gene expression system for Ustilago maydis. Obstacles of premature polyadenylation of the native tetR gene, high basal activity of the tetracycline-responsive promoter, and toxicity of the viral activation domain were overcome by designing a synthetic tetR* gene according to context-dependent codon usage, removing cryptic enhancer elements from the promoter, and using an acidic minimal activation domain, respectively. We verified tetracycline-dependent dose-response using optimised components and applied a straightforward single-step promoter replacement cassette to regulate expression of pheromone response factor, a key transcription factor regulating mating. Pheromone response in liquid culture and mating on solid media was abolished in the presence of tetracycline and doxycycline. Thus, functionality of this versatile new tool for the plant pathogen was proven in a biological context.

The phytopathogenic basidiomycete Ustilago maydis has become a model system for the analysis of plant-pathogen interactions. The genome sequence of this organism will soon be available, increasing the need for techniques to analyse gene function on a broad basis. We describe a heterologous transposition system for U. maydis that is based on the Caenorhabditis transposon Tc1, which is known to function independently of host factors and to be active in evolutionarily distant species. We have established a nitrate reductase based two-component counterselection system to screen for Tc1 transposition. The element was shown to be functional and transposed to several different locations in the genome of U. maydis. The insertion pattern observed was consistent with the proposed general mechanism of Tc1/mariner integration and constitutes a proof of principle for the first heterologous transposition system in a basidiomycete species. By mapping the insertion site context to known genomic sequences, Tc1 insertion events were shown to occur on different chromosomes, but exhibit a preference for non-coding regions. Only 20% of the insertions were found in putative open reading frames. The establishment of this system will permit efficient gene tagging in U. maydis and possibly also in other fungi.

In the corn smut fungus Ustilago maydis, pathogenic development is controlled by the b mating type locus that encodes the two homeodomain proteins bE and bW. A heterodimer of bE and bW controls a large set of genes, either directly by binding to cis regulatory sequences or indirectly via a b-dependent regulatory cascade. It is thought that several of the b-regulated genes contribute to processes involved in pathogenicity. In a screen for components of the b-dependent regulatory cascade we have isolated Hda1, a protein with homology to histone deacetylases of the RPD3 class. Hda1 can substitute for the histone deacetylase RPD3 in Saccharomyces cerevisiae, showing that it functions as a histone deacetylase. Deletion of hda1 results in the expression of several genes that are normally expressed only in the dikaryon, among these are several genes that are now expressed independently from their activation by the bE/bW heterodimer. hda1 mutant strains are capable to infect corn, and the proliferation of dikaryotic hyphae within the plant appears comparable to wild-type strains during initial developmental stages. Upon karyogamy, however, the proliferation to mature teliospores is blocked. The block in sporogenesis in Deltahda1 strains is probably a result of the deregulation of a specific set of genes whose temporal or spatial expression prevent the proper developmental progress.

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

In the corn smut fungus Ustilago maydis, pathogenic development is controlled by the b mating type locus that encodes the two homeodomain proteins bE and bW. A heterodimer of bE and bW controls a large set of genes, either directly by binding to cis regulatory sequences or indirectly via a b-dependent regulatory cascade. It is thought that several of the b-regulated genes contribute to processes involved in pathogenicity. In a screen for components of the b-dependent regulatory cascade we have isolated Hda1, a protein with homology to histone deacetylases of the RPD3 class. Hda1 can substitute for the histone deacetylase RPD3 in Saccharomyces cerevisiae, showing that it functions as a histone deacetylase. Deletion of hda1 results in the expression of several genes that are normally expressed only in the dikaryon, among these are several genes that are now expressed independently from their activation by the bE/bW heterodimer. hda1 mutant strains are capable to infect corn, and the proliferation of dikaryotic hyphae within the plant appears comparable to wild-type strains during initial developmental stages. Upon karyogamy, however, the proliferation to mature teliospores is blocked. The block in sporogenesis in Deltahda1 strains is probably a result of the deregulation of a specific set of genes whose temporal or spatial expression prevent the proper developmental progress.