José Pérez‐Martín

Ustilago maydis is an important fungal pathogen of maize, causing corn smut. It is well adapted to its host and proliferates in living plant tissue without inducing a defence response. The genome sequence of U. maydis has now been determined, the first for a biotrophic plant parasite. Several gene clusters that encode secreted proteins of unknown function were identified: genome-wide expression analysis shows that the clustered genes are upregulated during disease. Mutations in these gene clusters frequently affect virulence, ranging from complete loss of pathogenicity to hypervirulence. Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant–microbe interactions1. 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 development2. 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 factors3 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.

It is generally accepted that iron is the most important micronutrient used by bacteria. With members of the family Lactobacillae being the only exceptions so far (3), this metal is essential for cellular metabolism, since it is needed as a cofactor for a large number of enzymes (96). However, this element is not easily available to microorganisms in aerobic environments. While in anaerobic conditions Fe2+ is soluble at physiological pH and cells obtain iron without much difficulty from the external medium, the ion becomes quickly converted to Fe3+ upon exposure to oxygen and forms insoluble hydroxides at neutral pH, making the available metal very scarce (20). In order to acquire iron from the extracellular medium, virtually all aerobic bacteria produce and secrete low-molecular-weight compounds termed siderophores (sideros phoros, iron carriers). These compounds chelate Fe3+ with high affinity and specificity (68). Subsequently, the cell recovers the ferrisiderophore complexes through specific outer membrane receptors (30). Some of these high-affinity systems of iron uptake are important virulence factors in bacteria infecting animal fluids and tissues because they can chelate the metal bound to host proteins (7, 36, 60, 71). Furthermore, because iron availability is generally growth limiting for bacteria thriving in an animal millieu, the lack of the metal is a major environmental signal to trigger expression of virulence determinants (60). However, an excess of iron is toxic because of its ability to catalyse Fenton reactions and formation of active species of oxygen. Iron uptake has to be, therefore, exquisitely regulated to maintain the intracellular concentration of the metal between desirable limits. Considering that excretion mechanisms for iron are not known in bacteria, microorganisms appear to control iron homeostasis, regulating its transport through the membrane (5, 21).

The early notion of DNA as a passive target for regulatory proteins has given way to the realization that higher-order DNA structures and DNA-protein complexes are at the basis of many molecular processes, including control of promoter activity. Protein binding may direct the bending of an otherwise linear DNA, exacerbate the angle of an intrinsic bend, or assist the directional flexibility of certain sequences within prokaryotic promoters. The important, sometimes essential role of intrinsic or protein-induced DNA bending in transcriptional regulation has become evident in virtually every system examined. As discussed throughout this article, not every function of DNA bends is understood, but their presence has been detected in a wide variety of bacterial promoters subjected to positive or negative control. Nonlinear DNA structures facilitate and even determine proximal and distal DNA-protein and protein-protein contacts involved in the various steps leading to transcription initiation.

This review attempts to substantiate the notion that nonlinear DNA structures allow prokaryotic cells to evolve complex signal integration devices that, to some extent, parallel the transduction cascades employed by higher organisms to control cell growth and differentiation. Regulatory cascades allow the possibility of inserting additional checks, either positive or negative, in every step of the process. In this context, the major consequence of DNA bending in transcription is that promoter geometry becomes a key regulatory element. By using DNA bending, bacteria afford multiple metabolic control levels simply through alteration of promoter architecture, so that positive signals favor an optimal constellation of protein-protein and protein-DNA contacts required for activation. Additional effects of regulated DNA bending in prokaryotic promoters include the amplification and translation of small physiological signals into major transcriptional responses and the control of promoter specificity for cognate regulators.