José Pérez-Martín

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

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.

Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

Activation of the transcriptional regulator XylR of Pseudomonas putida by release of repression between functional domains

In the presence of toluene, xylenes and other structural analogues, the regulatory protein XylR, of the family of transcriptional regulators which act in concert with the σ54 factor, activate the promoter Pu of the TOL (toluene degradation) plasmid pWW0 of Pseudomonas putida. Amino acid changes Val‐219‐Asp and Ala‐220‐Pro, introducing a proline kink at the hinge region between the N‐terminal A domain and the central portion of XylR, resulted in a semi‐constitutive phenotype which mimicked the activating effect of aromatic inducers. This phenotype was further exacerbated by inserting extra amino acid residues within the same inter‐domain region. A truncated XylR protein devoid of the signal‐receiving, amino‐terminal portion of the protein stimulated the cognate promoter Pu at high levels independently of inducer addition, both in Escherichia coli and in Pseudomonas putida. Replacement of the amino‐terminal domain by a heterologous peptide derived from the MS2 virus polymerase resulted in a hybrid protein still able to bind DNA to the same extent in vivo as XylR, but unable to stimulate transcription. These data indicate that a key event in the activation of XyIR by toluene/xylenes is the release of the repression caused by the A domain of the protein on surfaces located at the central domain of the regulator.

The IIANtr (PtsN) Protein of Pseudomonas putida Mediates the C Source Inhibition of the ς54-dependent Pu Promoter of the TOL Plasmid

The gene cluster adjacent to the sequence of rpoN (encoding sigma factor ς54) ofPseudomonas putida has been studied with respect to the C source regulation of the Pu promoter of theupper TOL (toluene catabolism) operon. The region includes four open reading frames (ORFs), two of which (named ptsNand ptsO genes) encode proteins similar to components of the phosphoenolpyruvate:sugar phosphotransferase system. Each of the four genes was disrupted with a nonpolar insertion, and the effects in the inhibition caused by glucose on Pu activity were inspected with a lacZ reporter system. Although cells lacking ORF102, ORF284, and ptsO did not display any evident phenotype under the conditions tested, the loss ofptsN, which encodes the IIANtr protein, madePu unresponsive to repression by glucose. TheptsN mutant had rates of glucose/gluconate consumption identical to those of the wild type, thus ruling out indirect effects mediated by the transport of the carbohydrate. A site-directedptsN mutant in which the conserved phospho-acceptor site His68 of IIANtr was replaced by an aspartic acid residue made Pu blind to the presence or absence of glucose, thus supporting the notion that phosphorylation of IIANtr mediates the C source inhibition of the promoter. These data substantiate the existence of a molecular pathway for co-regulation of some ς54 promoters in which IIANtr is a key protein intermediate.

Dimorphism in fungal pathogens: Candida albicans and Ustilago maydis - similar inputs, different outputs

The ability to switch between a yeast-like form and a filamentous form is an extended characteristic among several fungi. In pathogenic fungi, this capacity has been correlated with virulence because along the infection process, dimorphic transitions are often required. Two well-known organisms for which dimorphism have been studied are the pathogenic fungi Candida albicans and Ustilago maydis, which infect mammals and corn, respectively. In both cases, several signal transduction pathways have been defined. Not surprisingly, these pathways are similar to the well-known pathways involved in the pseudohyphal differentiation that some Saccharomyces cerevisiae diploid strains show when nutrients are starved. However, in spite of similarities at the molecular level, strikingly, fungi use similar pathways to respond to environmental inputs, but with differing outcomes.

Regulatory noise in prokaryotic promoters: how bacteria learn to respond to novel environmental signals.

Various features of the regulation of pathways for biodegradation of recalcitrant compounds by Pseudomonas provide insights into the mechanisms by which operons evolve to acquire conditionally active promoters that permit the corresponding genes to be transcribed only when required. The regulatory noise hypothesis’proposes that transcriptional control systems develop responsiveness to new signals due to the leakiness and lack of specificity of preexisting promoters and regulators. When needed, these may become more specific through suppression of undesirable signals and further fine‐tuning of the recruited proteins to interact with distinct chemicals. This hypothesis is supported by the sophisticated regulation of α54‐dependent promoters of the TOL (toluene biodegradation) operons, which can be activated to various degrees by heterologous proteins. Such illegitimate’activation is suppressed by bent DNA structures, either static or protein induced, between promoter core elements. Therefore, not only the regulators but also the DNA sequences participate in the process that gives rise to novel specificities.

Pheromone-Induced G2 Arrest in the Phytopathogenic Fungus Ustilago maydis

ABSTRACT In the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydis cells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G2 phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.

ATP Binding to the σ54-Dependent Activator XylRTriggers a Protein Multimerization Cycle Catalyzed by UAS DNA

Abstract The events that take place at the prokaryotic enhancer of the Pu promoter of Pseudomonas putida prior to the engagement of the σ 54 -RNA polymerase (σ 54 -RNAP) have been studied in vitro. ATP hydrolysis by XylR, the cognate regulator of the system, is preceded by the multimerization of XylR at the enhancer, which is itself triggered by the sole allosteric effect of ATP binding to the protein. Since ADP is unable to support multimerization, ATP hydrolysis might be followed by a return to the nonmultimerized state. This notion is supported further by the properties of mutant proteins that seem to be frozen, in either the nonmultimerized or the multimerized state, respectively. These results support a cyclic mechanism of ATP-dependent association/dissociation of XylR at the promoter UAS that precedes any involvement of the polymerase in transcription initiation.

Ustilago maydis, a new fungal model system for cell biology

The use of fungal model systems, such as Saccharomyces cerevisisae and Schizosaccharomyces pombe, has contributed enormously to our understanding of essential cellular processes in animals. Here, we introduce the corn smut fungus Ustilago maydis as a new model organism for studying cell biological processes. Genome-wide analysis demonstrates that U. maydis is more closely related to humans than to budding yeast, and numerous proteins are shared only by U. maydis and Homo sapiens. Growing evidence suggests that basic principles of long-distance transport, mitosis and motor-based microtubule organization are conserved between U. maydis and humans. The fungus U. maydis, therefore, offers a unique system for the study of certain mammalian processes.

Involvement of σ54 in exponential silencing of the Pseudomonas putida TOL plasmid Pu promoter

The σ54‐dependent Pu promoter of the TOL plasmid pWWO of Pseudomonas putida becomes activated by the prokaryotic enhancer‐binding XyIR protein when cells encounter m‐xylene in the medium. However, even in the presence of the aromatic inducer, Pu activity is silenced in vivo during rapid exponential growth of the cells in rich medium. Various elements known to be involved in the control of the transcriptional activity of the promoter were examined to ascertain the mechanism by which expression of Pu is limited during the exponential phase of growth. A truncated and fully constitutive XyIR derivative deleted of its signal reception N‐terminal domain was found to be subjected to the same exponential silencing as the wild‐type XyIR when exposed to m‐xylene. This indicated that the phenomenon is not due to a late activation of XyIR by the aromatic effector. A Pu variant in which the integration host factor (IHF)‐binding site had been functionally replaced by a statically curved DNA segment showed the same induction pattern, thus ruling out variations in the intracellular levels of IHF changes during growth as the element responsible for the inactivity of Pu in rapidly growing cells. On the contrary, overproduction of the σ54 factor allowed Pu expression during exponential phase. As σ54 protein levels remained approximately constant during growth, the exponential silencing of Pu could be caused ultimately by changes in the activity of the factor itself. This effect may not be exclusive to Pu, but could be a general co‐regulation mechanism in σ54‐dependent promoters that connects transcription of a specific set of genes with the general physiological status of the cells.