Genetic analysis of biosurfactant production in Ustilago maydis

The dimorphic basidiomycete Ustilago maydis produces large amounts of surface-active compounds under conditions of nitrogen starvation. These biosurfactants consist of derivatives of two classes of amphipathic glycolipids. Ustilagic acids are cellobiose lipids in which the disaccharide is O-glycosidically linked to 15,16-dihydroxyhexadecanoic acid. Ustilipids are mannosylerythritol lipids derived from acylated beta-d-mannopyranosyl-d-erythritol. Whereas the chemical structure of these biosurfactants has been determined, the genetic basis for their biosynthesis and regulation is largely unknown. Here we report the first identification of two genes, emt1 and cyp1, that are essential for the production of fungal extracellular glycolipids. emt1 is required for mannosylerythritol lipid production and codes for a protein with similarity to prokaryotic glycosyltransferases involved in the biosynthesis of macrolide antibiotics. We suggest that Emt1 catalyzes the synthesis of mannosyl-d-erythritol by transfer of GDP-mannose. Deletion of the gene cyp1 resulted in complete loss of ustilagic acid production. Cyp1 encodes a cytochrome P450 monooxygenase which is highly related to a family of plant fatty acid hydroxylases. Therefore we assume that Cyp1 is directly involved in the biosynthesis of the unusual 15,16-dihydroxyhexadecanoic acid. We could show that mannosylerythritol lipid production is responsible for hemolytic activity on blood agar, whereas ustilagic acid secretion is required for long-range pheromone recognition. The mutants described here allow for the first time a genetic analysis of glycolipid production in fungi.     

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.     

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.     

Genetic diversity of Ustilago maydis strains

Thirty wild isolates belonging to five different locations in Mexico plus two laboratory strains of Ustilago maydis were characterized by restriction fragment length polymorphism (RFLP) analysis using 23 different clones as probes derived from a PstI library and two restriction enzymes. All loci analysed presented a high level of polymorphism, including one locus with thirty one different alleles. Geographical grouping of the populations was based on Nei's genetic distance and there was no correlation between genetic and geographic distances among these isolates. Our results suggest that DNA fingerprinting is a useful method for detecting genetic variation in populations of U. maydis. This work demonstrated that considerable genetic variation may be present within field populations of U. maydis.     

Ustilago maydis, the delightful blight.

Recent studies of the corn smut fungus life cycle and its regulation by two mating type loci and other genes provide a cornucopia of challenges in cell biology, genetics and protein structure. The fungus can exist in two states: nonpathogenic and pathogenic. The change from one state to the other is accompanied by a change in morphology (yeast-like to filamentous) and growth properties (saprophytic to parasitic).     

Endocytosis in the plant-pathogenic fungus Ustilago maydis

Summary. Filamentous fungi are an important group of tip-growing organisms, which include numerous plant pathogens such as Magnaporthe grisea and Ustilago maydis. Despite their ecological and economical relevance, we are just beginning to unravel the importance of endocytosis in filamentous fungi. Most evidence for endocytosis in filamentous fungi is based on the use of endocytic tracer dyes that are taken up into the cell and delivered to the vacuole. Moreover, genomewide screening for candidate genes in Neurospora crassa and U. maydis confirmed the presence of most components of the endocytic machinery, indicating that endocytosis participates in filamentous growth. Indeed, it was shown that in U. maydis early endosomes cluster at sites of growth, where they support morphogenesis and polar growth, most likely via endosome-based membrane recycling. In humans, such recycling processes to the plasma membrane involve small GTPases such as Rab4. A homologue of this protein is encoded in the genome of U. maydis but is absent from the yeast Saccharomyces cerevisiae, suggesting that Rab4-mediated recycling is important for filamentous growth. Furthermore, human Rab4 regulates traffic of early endosomes along microtubules, and a similar microtubule-based transport is described for U. maydis. These observations suggest that Rab4-like GTPases might regulate endosome- and microtubule-based recycling during tip growth of filamentous fungi. Correspondence and reprints: MPI für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Federal Republic of Germany.     

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.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.     

Inbreeding levels of two Ustilago maydis populations

Little is known about the population mating behavior of the smut fungus Ustilago maydis DC (Corda). To determine the amount of inbreeding that occurs in local U. maydis populations, two cornfields were sampled, one in North America (NA) at Le Sueur, Minnesota, and one in South America (SA) at Tarariras, Uruguay. These fields were chosen because of their geographic isolation and host management differences. Inbreeding coefficients (F(is)) were calculated using data derived from amplified fragment length polymorphism (AFLP) markers. Mean F(is) values estimated for both the NA (-0.08), and the SA (-0.02) populations statistically are not different from zero. The results of this study demonstrate that the U. maydis population structure in both cornfields results predominately from out-crossing and suggests that teliospores infrequently act as single infection units. The genetic differentiation between populations was high (F(st) = 0.25).     

Ustilago maydis secondary metabolism-from genomics to biochemistry

The dimorphic phytopathogenic fungus Ustilago maydis encounters different environments during its life cycle. As free-living unicellular haploid cell, the fungus must compete with other microorganisms for space and nutrients. As a pathogen, it also has to withstand the defense reactions of its host plant corn and to subvert the plant metabolism for its own purposes. During these interactions small molecules produced by the fungus serve important functions in the communication with its host and other organisms. The genome sequence of U. maydis makes it possible to deduce the full inventory of enzymatic functions that are involved in the production of these secondary metabolites. Although the fungus is known to secrete interesting small molecules the genome contains surprisingly few genes involved in the biosynthesis of polyketides (PKS) and non-ribosomal peptide synthetases (NRPS). Additional genes predicted to be part of secondary metabolism are located in subtelomeric regions suggesting that they are subject to high genetic and genomic variation. Here we review the pathways for the production of extracellular glycolipids that serve as biosurfactants, iron-chelating siderophores, tryptophan-derived indole pigments and indole acetic acid, the elucidation of which has greatly profited from the availability of the U. maydis genome sequence.     

Regulation of Nitrate Reductase in the Basidiomycete Ustilago maydis

Nitrate reductase was induced in Ustilago maydis by growth in medium containing only nitrate as the nitrogen source. Ammonium ions repressed the enzyme and led to a rapid loss of activity. Ammonium did not inhibit the enzyme in vitro; although amino acids partially did so, this cannot account for the rapid loss of in vivo activity which occurred when the ammonium was added. Experiments with cycloheximide and actinomycin D, together with measurements of protein turnover, suggested that nitrate reductase is actively broken down when cells with fully induced activity are transferred to medium containing ammonium ions.     

Cyclic AMP-dependent protein kinase from Ustilago maydis

Summary Ustilago maydis was surveyed for cyclic AMP-dependent protein kinase activity. Using a combination of ion-exchange and molecular filtration techniques, we demonstrate that there is only one form of cyclic AMP-dependent protein kinase in the cytosolic fraction of the fungus. The kinase activity is specifically activated by cyclic AMP and utilizes protamine and kemptide as substrates. Most, if not all, of the cyclic AMP binding detected in the soluble fraction is associated with the protein kinase activity. Cyclic AMP-dependent protein kinase is completely dissociated by cyclic AMP into catalytic and regulatory subunits having an apparent molecular weight of 35 000 daltons as judged by sucrose gradient centrifugation.Post graduate fellow from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina).Career investigator from CONICET.     

Towards understanding the extreme radiation resistance of Ustilago maydis

Ustilago maydis is a phytopathogenic fungus exhibiting extreme resistance to UV and ionizing radiation. The molecular mechanisms underlying this resistance are as yet unknown. The recently determined genome sequence was examined for clues to the radiation resistance, focusing on proteins in homologous recombination, but there was little that was unusual about them. Furthermore, by comparison, its recombinational repair system seems to be only minimally related to the extended synthesis-dependent DNA strand-annealing system of Deinococcus radiodurans. Thus, consideration should be given to the possibility that incremental structural changes in repair proteins or their elevated expression are the basis for the extreme radiation resistance in U. maydis. Evolution of a system enabling the survival of U. maydis under such conditions could be a secondary consequence of adaptation to an environment of continual genotoxic stress encountered in its habitat.     

The generic position of Ustilago maydis, Ustilago scitaminea, and Ustilago esculenta (Ustilaginales)

Three species of smut fungi (Ustilaginales, Basidiomycota) of economic importance, Ustilago maydis on corn, U. scitaminea on sugar cane, and U. esculenta on Zizania latifolia, were investigated in order to define their systematic position using morphological characteristics of the sori, ultrastructure of teliospore walls, and molecular data of the LSU rDNA. LSU rDNA suggests that U. maydis and U. scitaminea belong to the genus Sporisorium. This has already been proposed for U. scitaminea, which develops sori with whip-shaped axes corresponding to columellae. U. maydis and U. scitaminea, like typical species of Sporisorium, present peridia and columellae in their sori. Therefore, U. scitaminea is called Sporisorium scitamineum. U. maydis, however, is not placed in the genus Sporisorium here, because ongoing investigation of molecular data from the ITS rDNA region yields contradictory results and because the name Sporisorium maydis is occupied by an imperfect fungus. U. esculenta is recognized as Yenia esculenta. This placement in a separate genus is based on molecular data and on unique teliospore ultrastructure, i.e. apically enlarged, partly confluent warts developing on a strongly folded plasmalemma, and the exosporium and endosporium forming part of the ornamentation. Part 193 in the series „Studies in Heterobasidiomycetes“ from the Botanical Institute, University of Tübingen.