Genetic Control of Resistance to the Piperidine Fungicide Fenpropidin in Ustilago maydis

Mutants of Ustilago maydis (DC.) Corda, resistant to the piperidine fungicide fenpropidin, were isolated in a mutation frequency of 3.2 × 10^–5, after UV‐irradiation and selection on media containing 75 μg/ml fenpropidin. Genetic analysis with 15 such mutant isolates resulted in the identification of two unlinked chromosomal loci, U/fpd‐1 and U/fpd‐2. The U/fpd mutations are responsible for moderate resistance levels to fenpropidin (Rf: 42–56 or 15 based on effective concentration causing a 50% reduction in the growth rate (EC₅₀) or minimal inhibitory concentration (MIC) values, respectively). Haploid strains carrying both U/fpd mutations do not exhibit higher levels of resistance to fenpropidin, indicating no additivity of gene effect between non‐allelic genes. Cross‐resistance studies with other Sterol Biosynthesis inhibitors (SBIs) showed that the U/fpd‐mutant isolates exhibited a positive cross‐resistance to the piperidine piperalin and to the related morpholine fungicides fenpropimorph and tridemorph, but not to the inhibitors of C‐14 demethylase and squalene epoxidase. Crosses between mutants carrying the U/fpd‐genes with compatible isolates carrying the U/fpm or U/tdm mutations, which have been identified in previous genetic studies for resistance to morpholine fungicides fenpropimorph and tridemorph, yielded, with the exception of U/fpd‐2 × U/fpm‐2 crosses, a large number of recombinants with wild‐type sensitivity, indicating that the mutant genes involved were not allelic. Analysis of progeny from crosses between U/fpd‐2 and U/fpm‐2 mutants yielded no recombinants with wild‐type sensitivity, but a 1 : 1 progeny segregation was observed at the MIC for the U/fpd‐2 isolates, indicating that these genes are alleles of the same locus. A study of the fitness of fenpropidin‐resistant isolates showed that the U/fpd mutations do not affect the phytopathogenic fitness‐determining characteristics such as growth in liquid culture and pathogenicity on young corn plants.     

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.     

Mode of Action and Genetic Analysis of Resistance to Fluazinam in Ustilago maydis

Ustilago maydis strains, with low to moderate resistance to fluazinam (Rf ranging from 11.8 to 80), were isolated in a mutation frequency of 0.75 × 10⁻⁷ after chemical mutagenesis with N‐methyl‐N‐nitro‐N‐nitrosoguanidine (MNNG). Genetic analysis resulted in the identification of two chromosomal genes. A study of the effect of mutant genes in the phytopathogenic fitness of U. maydis revealed that the resistance mutations had no apparent effect on mycelia growth rate and pathogenicity on young corn plants. Cross‐resistant studies showed that the mutations for resistance to fluazinam were also responsible for resistance to oligomycin, but not to dinitrophenol. A dose‐dependent inhibition of glucose oxidation in whole cells was observed by both fluazinam and oligomycin, and a complete inhibition was found at 40 μg/ml. The results obtained provide strong evidence that the mode of action of fluazinam consists of the inhibition the fungal cell's energy production process through direct inhibition of the ATP synthetase.     

Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis’ itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5–fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical.     

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.     

Oxygen transfer and scale-up in lysine production by Ustilago maydis mutant

The oxygen transfer coefficient estimated by both sulfite and dynamic methods and some of the rheological properties of fermentation broths derived from the batch cultivation of a mutant of U. maydis in a sugar cane juice substrate, are used in a scaling-up procedure on the basis of the power consumption per volume unit concept. The fluid was of the Binham plastic type; the Np-N_RE correlations showed that the modified Reynolds numbers of the flat-blade turbine impellers were low, near to or in the laminar region; the Na-Pg/P relations were established and then used in the calculation regardless of the geometrical dissimilarities of the vessels. A change of scale from 5 to 50 liters was calculated and operated keeping the power per volume value constant. Reproduction of lysine yields, 2.5–3.2 g/liter, was repeatedly reached in 8 successive runs.     

The a and b loci of Ustilago maydis hybridize with DNA sequences from other smut fungi.

The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-type genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating system in which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hyphae. Cloned sequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to this finding include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiae.     

Induction of phenylalanine ammonia-lyase activity by tryptophan in Ustilago maydis.

To understand the regulation of phenylalanine ammonia-lyase (PAL) activity in the corn smut fungus, Ustilago maydis, we examined the effects of different media, metabolic effectors (including aromatic amino acids), and environmental factors on induction and repression of PAL activity. PAL was detected only in cell extracts and not in the culture medium. U. maydis PAL is constitutively produced at a low level in all media tested but its regulation can be influenced by aromatic amino acids. L-Tryptophan (0.3 mM) induces PAL activity 3- to 5-fold but tryptophan analogs and tryptophan-related metabolites do not. The enzyme is most readily induced during the early stationary phase of growth and the induced activity remains relatively constant during stationary stage. No induction or inhibition of PAL activity was observed as a function of culture temperature, pH or light. PAL induction was repressed by glucose but not by its reaction product, t-cinnamic acid. Induction did not require de novo protein synthesis, suggesting that some form of post-translational protein modification or a metabolic effect may be involved. This study shows that the regulation of U. maydis PAL is very different from the patterns known for plants and other fungi.     

Establishment of compatibility in the Ustilago maydis/maize pathosystem

The fungus Ustilago maydis is a biotrophic pathogen parasitizing on maize. The most prominent symptoms of the disease are large tumors in which fungal proliferation and spore differentiation occur. In this study, we have analyzed early and late tumor stages by confocal microscopy. We show that fungal differentiation occurs both within plant cells as well as in cavities where huge aggregates of fungal mycelium develop. U. maydis is poorly equipped with plant CWDEs and we demonstrate by array analysis that the respective genes follow distinct expression profiles at early and late stages of tumor development. For the set of three genes coding for pectinolytic enzymes, deletion mutants were generated by gene replacement. Neither single nor triple mutants were affected in pathogenic development. Based on our studies, we consider it unlikely that U. maydis feeds on carbohydrates derived from the digestion of plant cell wall material, but uses its set of plant CWDEs for softening the cell wall structure as a prerequisite for in planta growth.