Tracks for traffic: microtubules in the plant pathogen Ustilago maydis

Pathogenic development of the corn smut fungus Ustilago maydis depends on the ability of the hypha to grow invasively. Extended hyphal growth and mitosis require microtubules, as revealed by recent studies on the microtubule cytoskeleton. Surprisingly, hyphal tip growth involves only two out of 10 kinesins. Kinesin-3 is responsible for tip-directed (anterograde) endosome motility of early endosomes, which are thought to support hyphal elongation by apical membrane recycling. In addition, kinesin-3, together with kinesin-1 and myosin-5, appear to deliver secretory vesicles to the hyphal tip. Kinesin-1 also affects endosome motility by targeting cytoplasmic dynein to microtubule plus ends. This plus-end localization of dynein is essential for cell body-directed (retrograde) endosome motility, but also allows force generation during spindle elongation in mitosis. Furthermore, kinesin-1 and dynein participate in the organization of the microtubule array, thereby building their own network of tracks for intracellular motility. The recent progress in understanding microtubule-based processes in U. maydis has revealed an unexpected complexity of motor functions essential for the virulence of this pathogen. Further studies on structural and regulatory requirements for motor activity should help identify novel targets for fungicide development.     

Structure-function analysis of lipopeptide pheromones from the plant pathogen Ustilago maydis

Mating of two haploid cells is a prerequisite for the successful infection of corn by the pathogenic fungus Ustilago maydis. Cell-cell recognition is mediated by small lipopeptide pheromones. Genes encoding pheromone precursors as well as pheromone receptors are located in the a mating type locus. Two pheromones are known, the tridecapeptide a1 and the nonapeptide a2, both of which contain an S-prenylated cysteine methyl ester at the C-terminus. It has previously been shown that synthetic pheromones are active in a biological test system. Here, we used the same assay to perform a detailed analysis of synthetic a1 and a2 pheromones. Testing of truncated derivatives of a1 and a2 revealed that in both cases the pheromone function is less sensitive to N-terminal than to C-terminal truncations. Replacement of each amino acid in the a1 pheromone by either alanine or the corresponding D-amino acids revealed that four positions are important for function: the two central glycines (positions 5 and 9), proline at position 7 and tyrosine at position 10. By introducing different naturally occurring as well as synthetic amino acids at position 10, we demonstrate that the presence of an aromatic side chain at this position is necessary for function. We propose a model in which a cis peptide bond at proline 7 favours the formation of a type II' beta turn of the a1 pheromone backbone with glycine 9 in position i+1 (where i refers to the first position of the beta turn). As a result, tyrosine 10, at position i+2 of the turn, would be highly exposed and could be inserted into a structurally well-defined binding pocket of the receptor. The latter may represent an important facet of receptor specificity.     

The secretome of the maize pathogen Ustilago maydis

Ustilago maydis establishes a biotrophic relationship with its host plant, i.e. plant cells stay alive despite massive fungal growth in infected tissue. The genome sequence has revealed that U. maydis is poorly equipped with plant cell wall degrading enzymes and uses novel secreted protein effectors as crucial determinants for biotrophic development. Many of these effector genes are clustered and differentially regulated during plant colonization. In this review, we analyze the secretome of U. maydis by differentiating between secreted enzymes, likely structural proteins of the fungal cell wall (excluding GPI-anchored proteins) as well as likely effectors with either apoplastic or cytoplasmic function. This classification is based on the presence of functional domains, general domain structure and cysteine pattern. In addition, we discuss possible functions of selected protein classes with a special focus on disease development.     

A genome-based analysis of amino acid metabolism in the biotrophic plant pathogen Ustilago maydis

Amino acid, nitrogen and sulfur metabolism play critical roles in the growth and development of fungal pathogens both in and outside of the host. The genome sequence of Ustilago maydis provides an opportunity for exploring these biochemical pathways by comparison to known gene sequences from other fungi. This approach was used to identify candidate genes for almost all enzymes required for amino acid biosynthesis and degradation, as well as the uptake and assimilation of nitrogen and sulfur. A number of differences were found between U. maydis and other basidiomycetes, and between basidiomycetes and ascomycetes in general. The use of genomics to explore central metabolic pathways may be of value in characterizing strict biotrophic pathogens like U. maydis that seem to derive a very limited set of nutrients from the host and thus must retain extensive biosynthetic capacity.     

ras2 Controls morphogenesis,pheromone response,and pathogenicity in the fungal pathogen Ustilago maydis

Ustilago maydis, a pathogen of maize, is a useful model for the analysis of mating, pathogenicity, and the morphological transition between budding and filamentous growth in fungi. As in other fungi, these processes are regulated by conserved signaling mechanisms, including the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAP kinase) pathway. A current challenge is to identify additional factors that lie downstream of the cAMP pathway and that influence morphogenesis in U. maydis. In this study, we identified suppressor mutations that restored budding growth to a constitutively filamentous mutant with a defect in the gene encoding a catalytic subunit of PKA. Complementation of one suppressor mutation unexpectedly identified the ras2 gene, which is predicted to encode a member of the well-conserved ras family of small GTP-binding proteins. Deletion of the ras2 gene in haploid cells altered cell morphology, eliminated pathogenicity on maize seedlings, and revealed a role in the production of aerial hyphae during mating. We also used an activated ras2 allele to demonstrate that Ras2 promotes pseudohyphal growth via a MAP kinase cascade involving the MAP kinase kinase Fuz7 and the MAP kinase Ubc3. Overall, our results reveal an additional level of crosstalk between the cAMP signaling pathway and a MAP kinase pathway influenced by Ras2.     

Activation of the cAMP pathway in Ustilago maydis reduces fungal proliferation and teliospore formation in plant tumors

In the corn smut fungus Ustilago maydis, mating of two haploid sporidia is a prerequisite for subsequent colonization of the host. Cyclic AMP (cAMP) and pheromone signals have been implicated in this developmental program. The cAMP pathway is also needed for subsequent fungal development in planta, as null mutants in any component of the pathway fail to form tumors. Here we show that moderate activation of the pathway conferred either by mutation in the Galpha subunit or by mutation in the regulatory subunit of the protein kinase A influences tumor morphology. In the resulting tumors, the amount of fungal material is drastically reduced and fungal development is arrested at the stage of sporogenic hyphae. We conclude that tight regulation of the cAMP pathway is crucial for fungal development within the plant but does not interfere with the tumor induction process.     

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.     

A class-V myosin required for mating, hyphal growth, and pathogenicity in the dimorphic plant pathogen Ustilago maydis

In the early stages of plant infection, yeast-like haploid sporidia of Ustilago maydis respond to pheromone secreted by compatible partners by forming conjugation tubes. These then fuse to generate a dikaryotic hypha that forms appressoria to penetrate the host plant. As a first step toward understanding the structural requirements for these transitions, we have identified myo5, which encodes a class-V myosin. Analysis of conditional and null mutants revealed that Myo5 plays nonessential roles in cytokinesis and morphogenesis in sporidia and is required for hyphal morphology. Consistent with a role in morphogenesis, a functional green fluorescent protein-Myo5 fusion protein localized to the bud tip and the hyphal apex as well as to the septa and the spore wall during later stages of infection. However, the loss of Myo5 did not affect the tip growth of hyphae and sporidia. By contrast, Myo5 was indispensable for conjugation tube formation. Furthermore, myo5 mutants were impaired in the perception of pheromones, which indicates a particular importance of Myo5 in the mating process. Consequently, few mutant hyphae were formed that penetrated the plant epidermis but did not continue invasive growth. These results indicate a crucial role of Myo5 in the morphogenesis, dimorphic switch, and pathogenicity of U. maydis.     

Reprogramming a maize plant: transcriptional and metabolic changes induced by the fungal biotroph Ustilago maydis

The fungal pathogen Ustilago maydis establishes a biotrophic relationship with its host plant maize (Zea mays). Hallmarks of the disease are large plant tumours in which fungal proliferation occurs. Previous studies suggested that classical defence pathways are not activated. Confocal microscopy, global expression profiling and metabolic profiling now shows that U. maydis is recognized early and triggers defence responses. Many of these early response genes are downregulated at later time points, whereas several genes associated with suppression of cell death are induced. The interplay between fungus and host involves changes in hormone signalling, induction of antioxidant and secondary metabolism, as well as the prevention of source leaf establishment. Our data provide novel insights into the complexity of a biotrophic interaction.     

Sending mixed signals: redundancy vs. uniqueness of signaling components in the plant pathogen, Ustilago maydis

The ability to respond to a changing environment separates successful organisms from their competitors. Thus, signal transduction is a crucial aspect of an organism's growth, development, differentiation, and reproduction. Nowhere is this more evident than in the co-evolution of obligate pathogens with their host organisms. The genome sequence of Ustilago maydis, the pathogen of maize, has provided a powerful tool in the assessment and characterization of signaling pathways for this organism. Inspection of the sequence reveals that while U. maydis has a streamlined gene content, it appears to contain a full repertoire of the standard signaling cascades present in other fungi. A full range of paralogues are present to provide redundancy of function on the one hand while, on the other, distinct strategies for survival. This review explores signaling based on the conserved mitogen-activated protein (MAP) kinase and cAMP-dependent protein kinase A (PKA) pathways as well as ancillary functions, with emphasis on the unique aspects of the U. maydis approach to utilizing this architecture.     

Glycolysis in Ustilago maydis

The kinetic parameters of the 10 glycolytic enzymes and glycolytic fluxes were determined for the first time in Ustilago maydis. Enzyme activities in yeast grown in minimal medium and harvested in the stationary stage were twofold higher than those from yeast grown in rich medium. In contrast, in yeast harvested in the exponential stage, the enzyme activities were higher in cells grown in rich medium. Phosphofructokinase activity was the lowest in the four culture conditions analyzed, suggesting that this enzyme is a flux-controlling step in U. maydis glycolysis. The V(max) and K(m) values of hexokinase and pyruvate kinase were similar under all conditions. The results revealed that U. maydis aldolase belongs to the class II type of metalo-aldolases. 3-Phosphoglycerate mutase (PGAM) activity was 2,3-bisphosphoglycerate cofactor independent, which contrasted with the cofactor dependency predicted by the amino acid sequence alignment analysis. Pyruvate was secreted by U. maydis yeast in the presence and absence of external glucose. The glycolytic enzyme activities in the U. maydis mycelial form were similar to those found in yeast, except for one order of magnitude higher phosphofructokinase and PGAM activities, thus suggesting differences in the glycolysis regulatory mechanisms between the two cellular forms.