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.

Summary: An interesting observation, reported for transgenic plants that have been engineered to overproduce osmolytes, is that they often exhibit impaired growth in the absence of stress. As growth reduction and accumulation of osmolytes both typically result from adaptation, we hypothesized that growth reduction may actually result from osmolyte accumulation. To examine this possibility more closely, intracellular proline level was manipulated by expressing mutated derivatives of tomPRO2 (a Δ¹‐pyrroline‐5‐carboxylate synthetase, P5CS, from tomato) in Saccharomyces cerevisiae. This was done in the presence and absence of a functional proline oxidase, followed by selection and screening for increased accumulation of proline in the absence of any stress. Here we show, in support of our hypothesis, that the level of proline accumulation and the amount of growth are inversely correlated in cells grown under normal osmotic conditions. In addition, the intracellular concentration of proline also resulted in increases in ploidy level, vacuolation and altered accumulation of several different transcripts related to cell division and gene expression control. Because these cellular modifications are common responses to salt stress in both yeast and plants, we propose that proline and other osmolytes may act as a signaling/regulatory molecule able to activate multiple responses that are part of the adaptation process. As in previous studies with transgenic plants that overaccumulate osmolytes, we observed some increase in relative growth of proline‐overaccumulating cells in mild hyperosmotic stress.

Summary: Programmed cell death (PCD) is a fundamental cellular process conserved in metazoans, plants and yeast. Evidence is presented that salt induces PCD in yeast and plants because of an ionic, rather than osmotic, etiology. In yeast, NaCl inhibited growth and caused a time‐dependent reduction in viability that was preceded by DNA fragmentation. NaCl also induced the cytological hallmarks of lysigenous‐type PCD, including nuclear fragmentation, vacuolation and lysis. The human anti‐apoptotic protein Bcl‐2 increased salt tolerance of wild‐type yeast strain and calcineurin‐deficient yeast mutant (cnb1Δ) that is defective for ion homeostasis, but had no effect on the NaCl or sorbitol sensitivity of the osmotic hypersensitive hog1Δ mutant – results that further link PCD in the response to the ion disequilibrium under salt stress. Bcl‐2 suppression of cnb1Δ salt sensitivity was ENA1 (P‐type ATPase gene)‐dependent, due in part to transcriptional activation. Salt‐induced PCD (TUNEL staining and DNA laddering) in primary roots of both Arabidopsis thaliana wild type (Col‐1 gl1) and sos1 (salt overly sensitive) mutant seedlings correlated positively with treatment lethality. Wild‐type plants survived salt stress levels that were lethal to sos1 plants because secondary roots were produced from the shoot/root transition zone. PCD‐mediated elimination of the primary root in response to salt shock appears to be an adaptive mechanism that facilitates the production of roots more able to cope with a saline environment. Both salt‐sensitive mutants of yeast (cnb1Δ) and Arabidopsis (sos1) exhibit substantially more profound PCD symptoms, indicating that salt‐induced PCD is mediated by ion disequilibrium.

In nature,Saccharomycesyeasts manifest a number of adaptive responses to overcome adverse environments such as filamentation, invasive growth, flocculation and adherence to solid surfaces. CertainSaccharomyceswild yeasts, namely “flor yeasts,” have also acquired the ability to form a buoyant biofilm at the broth surface. Here we report that mutations in a single gene, identified asFLO11, separate these “floating” yeasts from their nonfloating relatives. We have determined that the capability to form a self-supporting biofilm at the liquid surface is largely dependent on two changes in theFLO11gene. First, we identified a 111-nt deletion within a repression region of theFLO11promoter that significantly increasesFLO11gene expression. Secondly, we found rearrangements within the central tandem repeat domain of the coding region that yield a more hydrophobic Flo11p variant. Together, these mutations result in dramatic increase in cell surface hydrophobicity, which in turn confers these yeasts the ability to float by surface tension, an adaptive mechanism to gain direct access to oxygen within oxygen-poor liquid environments.