The Resistance of Maize to Ustilago maydis Infection Is Correlated with the Degree of Methyl Esterification of Pectin in the Cell Wall.

Common smut caused by Ustilago maydis is one of the dominant fungal diseases in plants. The resistance mechanism to U. maydis infection involving alterations in the cell wall is poorly studied. In this study, the resistant single segment substitution line (SSSL) R445 and its susceptible recurrent parent line Ye478 of maize were infected with U. maydis, and the changes in cell wall components and structure were studied at 0, 2, 4, 8, and 12 days postinfection. In R445 and Ye478, the contents of cellulose, hemicellulose, pectin, and lignin increased by varying degrees, and pectin methylesterase (PME) activity increased. The changes in hemicellulose and pectin in the cell wall after U. maydis infection were analyzed via immunolabeling using monoclonal antibodies against hemicellulsic xylans and high/low-methylated pectin. U. maydis infection altered methyl esterification of pectin, and the degree of methyl esterification was correlated with the resistance of maize to U. maydis. Furthermore, the relationship between methyl esterification of pectin and host resistance was validated using 15 maize inbred lines with different resistance levels. The results revealed that cell wall components, particularly pectin, were important factors affecting the colonization and propagation of U. maydis in maize, and methyl esterification of pectin played a role in the resistance of maize to U. maydis infection.

Co-immunoprecipitation-Based Identification of Effector-Host Protein Interactions from Pathogen-Infected Plant Tissue.

Protein-protein interactions play an essential role in host-pathogen interactions. Phytopathogens secrete a cocktail of effector proteins to suppress plant immunity and reprogram host cell metabolism in their favor. Identification and characterization of effectors and their target protein complexes by co-immunoprecipitation can help to gain a deeper understanding of the functions of individual effectors during pathogenicity and can also provide new insights into the wiring of plant signaling pathways or metabolic complexes. Here we describe a detailed protocol to perform co-immunoprecipitation of effector-target protein complexes from plant extracts with an example of the Ustilago maydis/maize pathosystem for which we also provide a fungal protoplast transformation and maize seedling infection protocols.

Targeted transcriptomic study of the implication of central metabolic pathways in mannosylerythritol lipids biosynthesis in Pseudozyma antarctica T-34.

Pseudozyma antarctica is a nonpathogenic phyllosphere yeast known as an excellent producer of industrial lipases and mannosylerythritol lipids (MELs), which are multi-functional glycolipids. The fungus produces a much higher amount of MELs from vegetable oil than from glucose, whereas its close relative, Ustilago maydis UM521, produces a lower amount of MELs from vegetable oil. In the present study, we used previous gene expression profiles measured by DNA microarray analyses after culturing on two carbon sources, glucose and soybean oil, to further characterize MEL biosynthesis in P. antarctica T-34. A total of 264 genes were found with induction ratios and expression intensities under oily conditions with similar tendencies to those of MEL cluster genes. Of these, 93 were categorized as metabolic genes using the Eukaryotic Orthologous Groups classification. Within this metabolic category, amino acids, carbohydrates, inorganic ions, and secondary metabolite metabolism, as well as energy production and conversion, but not lipid metabolism, were enriched. Furthermore, genes involved in central metabolic pathways, such as glycolysis and the tricarboxylic acid cycle, were highly induced in P. antarctica T-34 under oily conditions, whereas they were suppressed in U. maydis UM521. These results suggest that the central metabolism of P. antarctica T-34 under oily conditions contributes to its excellent oil utilization and extracellular glycolipid production.

Complementing the intrinsic repertoire of Ustilago maydis for degradation of the pectin backbone polygalacturonic acid.

Microbial valorization of plant biomass is a key target in bioeconomy. A promising candidate for consolidated bioprocessing is the dimorphic fungus Ustilago maydis. It harbors hydrolytic enzymes to degrade biomass components and naturally produces valuable secondary metabolites like itaconic acid, malic acid or glycolipids. However, hydrolytic enzymes are mainly expressed in the hyphal form. This type of morphology should be prevented in industrial fermentation processes. Genetic activation of these enzymes can enable growth on cognate substrates also in the yeast form. Here, strains were engineered for growth on polygalacturonic acid as major component of pectin. Besides activation of intrinsic enzymes, supplementation with heterologous genes for potent enzymes was tested. The presence of an unconventional secretion pathway allowed exploiting fungal and bacterial enzymes. Growth of the engineered strains was evaluated by a recently developed method for online determination of residual substrates based on the respiration activity. This enabled the quantification of the overall consumed substrate as a key asset for the assessment of the enzyme degradation potential even on polymeric substrates. Co-fermentation of endo- and exo-polygalacturonase overexpression strains resulted in efficient growth on polygalacturonic acid. In the future, the approach will be extended to establish efficient degradation and valorization of pectin.

Heterologous expression and characterization of the aspartic endoprotease Pep4um from Ustilago maydis, a homolog of the human Chatepsin D, an important breast cancer therapeutic target.

The pep4um gene (um04926) of Ustilago maydis encodes a protein related to either vacuolar or lysosomal aspartic proteases. Bioinformatic analysis of the Pep4um protein revealed that it is a soluble protein with a signal peptide suggesting that it likely passes through the secretory pathway, and it has two probable self-activation sites, which are similar to those in Saccharomyces cerevisiae PrA. Moreover, the active site of the Pep4um has the two characteristic aspartic acid residues of aspartyl proteases. The pep4um gene was cloned, expressed in Pichia pastoris and a 54 kDa recombinant protein was observed. Pep4um-rec was confirmed to be an aspartic protease by specifically inhibiting its enzymatic activity with pepstatin A. Pep4um-rec enzymatic activity on acidic hemoglobin was optimal at pH 4.0 and at 40 °C. To the best of our knowledge this is the first report about the heterologous expression of an aspartic protease from a basidiomycete. An in-depth in silico analysis suggests that Pep4um is homolog of the human cathepsin D protein. Thus, the Pep4um-rec protein may be used to test inhibitors of human cathepsin D, an important breast cancer therapeutic target.

Aeroallergens in North-Central Nigeria.

INTRODUCTION AND OBJECTIVES: Aeroallergens are airborne organic substances which are responsible for allergenic diseases in hypersensitive individuals. People are exposed to their allergens either directly or after their entrance into the interiors. The spatio-temporal pattern of aeroallergens and their relationship with weather variability in Abuja and Nassarawa, North-Central Nigeria was studied. MATERIALS AND METHODS: Aerosamples were trapped with modified Tauber-like pollen traps. Samples were collected monthly and centrifuged at 2500rpm for 5 min and subjected to acetolysis. Meteorological data were collected from the Nigerian Meteorological Agency. RESULTS AND CONCLUSION: Aeroallergens concentration were unequivocally regulated by weather variables in both locations, indicating the possible use of aeroallergens especially pollen and spores as bio-indicators of weather variations and change. Aeroallergens encountered were fungal spores, pollen, diatom frustules, fern spores, algal cyst/cells in decreasing order of dominance. Among pollen group, Poaceae, Amarathaceae/Chenopodiaceae and Hymenocardia acida dominated. Spores of Smut species, Puccinia, Curvularia and Nigrospora were major contributors among aeromycoflora. Fungal spores morphotype dominated during the rainier months and were major contributors of the aeroallergen spectrum with those belonging to Deuteromycete preponderant. Aeroallergens which were previously identified as triggers of conjunctivitis, asthma, allergic sinusitis and bronchopulmonary allergic diseases were frequently present in both locations. Pollen prevailed more during the harmattan, influenced by northeast trade wind. Pollen component differed and was based on autochthonous source plants, indicating difference in sub-vegetational types.

Transcriptomic analysis of basidiocarp development in Ustilago maydis (DC) Cda.

Previously, we demonstrated that when Ustilago maydis (DC) Cda., a phytopathogenic basidiomycete and the causal agent of corn smut, is grown in the vicinity of maize embryogenic calli in a medium supplemented with the herbicide Dicamba, it developed gastroid-like basidiocarps. To elucidate the molecular mechanisms involved in the basidiocarp development by the fungus, we proceeded to analyze the transcriptome of the process, identifying a total of 2002 and 1064 differentially expressed genes at two developmental stages, young and mature basidiocarps, respectively. Function of these genes was analyzed with the use of different databases. MIPS analysis revealed that in the stage of young basidiocarp, among the ca. two thousand differentially expressed genes, there were some previously described for basidiocarp development in other fungal species. Additional elements that operated at this stage included, among others, genes encoding the transcription factors FOXO3, MIG3, PRO1, TEC1, copper and MFS transporters, and cytochromes P450. During mature basidiocarp development, important up-regulated genes included those encoding hydrophobins, laccases, and ferric reductase (FRE/NOX). The demonstration that a mapkk mutant was unable to form basidiocarps, indicated the importance of the MAPK signaling pathway in this developmental process.

Ustilago echinata: Infection in a Mixed Martial Artist Following an Open Fracture.

Ustilago, a common fungal parasite of grains, is infrequently isolated as a pathogen in humans. We describe a case of Ustilago echinata infection following an open distal tibia fracture, review the current literature of this genus as a cause of invasive fungal infection in humans, and discuss management issues.

Antibacterial Activities of Endophytic Fungi Isolated from Mentha cordifolia Leaves and Their Volatile Constituents.

A total of 17 endophytic fungal isolates were obtained from the leaves of Mentha cordifolia Opiz (Lamiaceae). Seven isolates were identified to the level of genus by using taxonomically relevant morphological traits. Colletotrichum and Phoinopsis species were dominant among these strains. All strains were separated from M cordifolia leaf for the first time. The ethyl acetate extracts of all endophytic fungi were tested for antibacterial activity against Salmonella typhimurium TISTR 1166 and Pseudomonas aeruginosa TISTR781. Most endophytes exhibited antibacterial activity. Ustilago sp. MFLUCC15-1024 presented the highest inhibition zone diameter with a MIC of 31.25 µg/mL against the tesfed pathogens. The chemical composition of the ethyl acetate extract of this strain was investigated using gas chromatography-mass spectrometry. Twenty-one components were identified. 2-Phenylethanol (38.7%), E-ligustilide (12.4%), a-eudesmol (10.2%), ß-vetivone (4.6%), ß-ylangene (3.7%) and verbanol (3.4%) were the major components of the extract. The strong antibacterial activity of Ustilago sp. MFLUCC15-1024 ethyl acetate extract may be attributed to the presence of a high concentration of bioactive compounds including phenyl ethyl alcohol, E-ligustilide and a-eudesmol. The results indicate that there is high diversity of endophytic fungi in M cordifolia leaf, and that Ustilago sp. MFLUCC l5-1024 strain could be an excellent resource of natural antibacterial compounds.

The 3-hydroxy-3-methylglutaryl coenzyme-A reductases from fungi: a proposal as a therapeutic target and as a study model.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) catalyzes the conversion of HMG-Co-A into mevalonate. This step is the limiting point for the synthesis of cholesterol in mammals and ergosterol in fungi. We describe in this article the genome organization of HMGR coding genes and those deduced from different fungi, recount the evidence showing statins as HMGR inhibitors for ergosterol synthesis and its effect in yeast viability, and propose fungal HMGR (HMGRf) as a model to study the use of pharmaceutical compounds to inhibit cholesterol and ergosterol synthesis. Bibliographical search and bioinformatic analyses were performed and discussed. HMGRfs belong to the class I with a high homology in the catalytic region. The sterol biosynthetic pathway in humans and fungi share many enzymes in the initial steps (such as the HMGR enzyme), but in the last steps enzymes are different rendering the two final products: cholesterol in mammals and ergosterol in fungi. With regards to inhibitors such as statins and other compounds, these affect also fungal viability. Since HMGR from Schizosaccharomyces pombe and Ustilago maydis are very similar to the human HMGR in the catalytic regions, we propose that fungal enzymes can be used to test inhibitors for a potential use in humans. We consider that HMGRf is a good therapeutic target to design and test new antifungal compounds. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Biological activity of sedaxane---a novel broad-spectrum fungicide for seed treatment.

BACKGROUND: Sedaxane is a new broad-spectrum seed treatment fungicide developed by Syngenta Crop Protection for control of seed- and soil-borne diseases in a broad range of crops. Its physicochemical properties and activity spectrum have been optimised for use as a seed treatment providing both local and systemic protection of the seed and roots of target crops. RESULTS: Sedaxane inhibits respiration by binding to the succinate dehydrogenase complex in the fungal mitochondrium. Its activity spectrum covers seed-borne fungi such as Ustilago nuda, Tilletia caries, Monographella nivalis and Pyrenophora graminea, as well as the soil-borne fungi Rhizoctonia solani, R. cerealis and Typhula incarnata. Under greenhouse conditions, sedaxane showed high levels and consistent protection against U. nuda, P. graminea and Rhizoctonia spp. Under field conditions, efficacy against Rhizoctonia spp. resulted in increased yield compared with the untreated check. Efficacy against snow mould has been shown under very high disease pressure conditions. The combination of sedaxane plus fludioxonil against snow mould can provide resistance management for sustainable use. CONCLUSIONS: The broad spectrum and high level of activity in combination with excellent crop tolerance allow the use of sedaxane as a seed treatment in a wide variety of crops. It is a potential tool for precautionary resistance management when combined with other fungicides, especially against pathogens showing a potential for resistance development, such as M. nivalis.

Impact of applying edible oils to silk channels on ear pests of sweet corn.

The impact of applying edible oils to corn silks on ear-feeding insects in sweet corn, Zea mays L., production was evaluated in 2006 and 2007. Six edible oils used in this experiment were canola, corn, olive, peanut, sesame, and soybean. Water and two commercial insecticidal oils (Neemix neem oil and nC21 Sunspray Ultrafine, a horticultural mineral oil) were used as the controls for the experiment. Six parameters evaluated in this experiment were corn earworm [Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae)] damage rating, the number of sap beetle [Carpophilus spp. (Coleoptera: Nitidulidae)] adults and larvae, the number of corn silk fly (or picture-winged fly) (Diptera: Ulidiidae) larvae, common smut [Ustilago maydis (D.C.) Corda] infection rate, and corn husk coverage. Among the two control treatments, neem oil reduced corn earworm damage at both pre- and postpollination applications in 2006, but not in 2007, whereas the mineral oil applied at postpollination treatments reduced corn earworm damage in both years. The mineral oil also reduced the number of sap beetle adults, whereas the neem oil applied at postpollination attracted the most sap beetle adults in 2007. Among the six edible oil treatments, the corn and sesame oils applied at postpollination reduced corn earworm damage only in 2007. The application of the peanut oil at postpollination attracted more sap beetle adults in 2006, and more sap beetle larvae in 2007. Olive and neem oils significantly reduced husk coverage compared with the water control in both years. The mineral oil application consistently increased smut infection rate in both 2006 and 2007. Ramifications of using oil treatments in ear pest management also are discussed.

Bioconversion of crude glycerol to glycolipids in Ustilago maydis.

Ustilago maydis is known to produce glycolipid-type biosurfactants. Here, we show that U. maydis is able to efficiently convert biodiesel-derived crude glycerol to glycolipids. We have optimized the medium composition and environmental factors for bioconversion of crude glycerol to glycolipids. The synthetic medium (MinCG) contains 50 g L(-1) crude glycerol and 20.3 mg L(-1) ammonium citrate as the carbon and nitrogen sources, respectively. The supplementation of trace amount of amino acids, Group-B vitamins and precursors of glycolipids, mannose and erythritol, also improved the final yield. At pH 4.0 and 30°C, 32.1 g L(-1) total glycolipids was produced in a 8.2-day fed-batch bioprocess. Methanol at 2% or above severely inhibited cell growth and production of glycolipids. Our results suggest that U. maydis is an excellent host for the bioconversion of crude glycerol to value-added products.

Homology modeling and screening of new 14α-demethylase inhibitor (DMI) fungicides based on optimized expression of CYP51 from Ustilago maydis in Escherichia coli.

Ustilago maydis infection is a serious disease affecting corn crops worldwide. Sterol 14α-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis and an effective target of antifungal drugs. To further study the interaction between CYP51 and drugs and exploit more specific 14α-demethylase inhibitor (DMI) fungicides for U. maydis, in this study homology modeling of CYP51 from U. maydis (UmCYP51) templated as the eukaryotic orthologues (the human CYP51) and screening of new DMI fungicides based on optimized expression were carried out for the first time. In addition, XF-113 and ZST-4 were screened by analyzing the spectral characteristics between the purified UmCYP51-35 and fungicides. These results provide a theoretical basis and new ideas for efficient design and development of new antifungal drugs.

The snf1 gene of Ustilago maydis acts as a dual regulator of cell wall degrading enzymes.

Many fungal plant pathogens are known to produce extracellular enzymes that degrade cell wall elements required for host penetration and infection. Due to gene redundancy, single gene deletions generally do not address the importance of these enzymes in pathogenicity. Cell wall degrading enzymes (CWDEs) in fungi are often subject to carbon catabolite repression at the transcriptional level such that, when glucose is available, CWDE-encoding genes, along with many other genes, are repressed. In Saccharomyces cerevisiae, one of the main players controlling this process is SNF1, which encodes a protein kinase. In this yeast, Snf1p is required to release glucose repression when this sugar is depleted from the growth medium. We have employed a reverse genetic approach to explore the role of the SNF1 ortholog as a potential regulator of CWDE gene expression in Ustilago maydis. We identified U. maydis snf1 and deleted it from the fungal genome. Consistent with our hypothesis, the relative expression of an endoglucanase and a pectinase was higher in the wild type than in the Δsnf1 mutant strain when glucose was depleted from the growth medium. However, when cells were grown in derepressive conditions, the relative expression of two xylanase genes was unexpectedly higher in the Δsnf1 strain than in the wild type, indicating that, in this case, snf1 negatively regulated the expression of these genes. Additionally, we found that, contrary to several other fungal species, U. maydis Snf1 was not required for utilization of alternative carbon sources. Also, unlike in ascomycete plant pathogens, deletion of snf1 did not profoundly affect virulence in U. maydis.

[Identification of disease resistance to Ustilago coicis in southern Coix germplasm].

OBJECTIVE: To identified the resistance of Coix to Ustilago coicis and screen the high disease-resistance Coix germplasm. METHOD: Field and laboratory biochemical methods were used for the resistance identification. Ninteen germplasms collected from 7 provinces in southern of China such as Yunnan, Zhejiang, Fujian etc. were inoculated with chlamydospore of U. coicis, respectively. The incidence of a disease in field was investigated and the level of resistance was evaluated. The PAL activity dynamic changes in different level resistant germplasms were further determined. RESULT: The result of field test showed 1 germplasm was immune, 1 germplasm was high resistance which incidence rate was under 20%, 6 germplasms were moderate resistance with the average incidence rates ranged within 20% - 40%, 11 of 19 germplasms that average incidence rates above 40% were identified as sensitive resistance. The value of PLA activity peak of resistant germplasm in seedling was significant higher and appeared earlier than that of the sensitive ones after inoculating. CONCLUSION: Most collected C. lacryma-jobi germplasms are sensitive to smut in our investigation; the PAL activity may play important role in Coix germplasm for resistance to smut and the biochemical method may be as an aiding method to resistance identification of Coix germplasm.

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.

Functional expression of human dihydroorotate dehydrogenase (DHODH) in pyr4 mutants of ustilago maydis allows target validation of DHODH inhibitors in vivo.

Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.

Osteoclast-forming suppressive compounds from Makomotake, Zizania latifolia infected with Ustilago esculenta.

A novel compound (1) and a known one (2) were isolated from Makomotake, Zizania latifolia infected with Ustilago esculenta, as osteoclast-forming suppressive substances.

Pollen and spores in the air of a hospital out-patient ward.

BACKGROUND: Aerobiological studies of interest to the allergy specialist are routinely carried out using information from outdoor traps. However, most of our time is spent indoors and it is often the content of this air that is responsible for allergic phenomena. MATERIAL AND METHODS: The air of a hospital outpatient ward was analyzed using two portable volumetric aerobiological traps, one at floor level and the other at a height of 1 meter. Both spores and pollen grains were counted and their values were compared with those outside the building. RESULTS AND CONCLUSIONS: Twenty types of pollen grains were found. Their concentrations ranged from 2.7 and 25.1 grains/m3, with the most frequent being (in order) grasses, evergreen oak (holm and cork oaks), water plantain, and olive. Twenty two different types of spores were found with concentrations of between 175.0 and 1395.8 spores/m3 and the most frequent were Cladosporium, Ustilago and basidiospores. No significant differences were found between the floor level and the meter-high measurements. Comparison with outdoor levels showed that the three most abundant pollen types were correlated, with a ratio of 30:1 between indoor and outdoor levels. For the spores and fungi propagules, the indoor:outdoor ratio was highly variable, depending on the type. Thus, for Aspergillus-Penicillium spores, the concentration was even higher indoors than outdoors, although for most types, lower levels were found indoors, with a mean outdoor:indoor ratio of 4:1. We argue that this relationship reflects the presence of indoor spore sources. Rainfall was correlated with a decline in pollen levels and a rise in spore levels.