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The phytopathogenic fungus Ustilago maydis causes corn smut by suppressing host plant defenses, including the oxidative burst response. While many studies have investigated how U. maydis responds to oxidative stress during infection, the consequences of heightened resistance to oxidative stress on virulence remain understudied. This study aimed to identify the effects on virulence in U. maydis strains exhibiting enhanced resistance to hydrogen peroxide (H 2 O 2 ).To achieve this, we exposed U. maydis SG200 to 20 escalating H 2 O 2 shocks, resulting in an adapted strain resistant to concentrations as high as 60 mM of H 2 O 2 , a lethal dose for the initial strain. Genetic analysis of the adapted strain revealed five nucleotide substitutions, two minor copy number variants, and a large amplification event on chromosome nine (1-149 kb) encompassing the sole catalase gene. Overexpressing catalase increased resistance to H 2 O 2 ; however, this resistance was lower than that observed in the adapted strain. Additionally, virulence was reduced in both strains with enhanced H 2 O 2 resistance.In summary, enhanced H 2 O 2 resistance, achieved through either continuous exposure to the oxidative agent or through catalase overexpression, decreased virulence. This suggests that the response to the oxidative stress burst in U. maydis is optimal and that increasing the resistance to H 2 O 2 does not translate into increased virulence. These findings illuminate the intricate relationship between oxidative stress resistance and virulence in U. maydis, offering insights into its infection mechanisms., (© 2024. The Author(s).)

Ustilago maydis encodes ten predicted light-sensing proteins. The biological functions of only a few of them are elucidated. Among the characterized ones are two DNA-photolyases and two rhodopsins that act as DNA-repair enzymes or green light-driven proton pumps, respectively. Here we report on the role of two other photoreceptors in U. maydis, namely White collar 1 (Wco1) and Phytochrome 1 (Phy1). We show that they bind flavins or biliverdin as chromophores, respectively. Both photoreceptors undergo a photocycle in vitro. Wco1 is the dominant blue light receptor in the saprophytic phase, controlling all of the 324 differentially expressed genes in blue light. U. maydis also responds to red and far-red light. However, the number of red or far-red light-controlled genes is less compared to blue light-regulated ones. Moreover, most of the red and far-red light-controlled genes not only depend on Phy1 but also on Wco1, indicating partial coregulation of gene expression by both photoreceptors. GFP-fused Wco1 is preferentially located in the nucleus, Phy1 in the cytosol, thus providing no hint that these photoreceptors directly interact or operate within the same complex. This is the first report on a functional characterization and coaction of White collar 1 and phytochrome orthologs in basidiomycetes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The evolutionarily conserved serine/threonine kinase TOR recruits different subunits to assemble the Target of Rapamycin Complex 1 (TORC1), which is inhibited by rapamycin and regulates ribosome biogenesis, autophagy, and lipid metabolism by regulating the expression of lipogenic genes. In addition, TORC1 participates in the cell cycle, increasing the length of the G2 phase. In the present work, we investigated the effect of rapamycin on cell growth, cell morphology and neutral lipid metabolism in the phytopathogenic fungus Ustilago maydis. Inhibition of TORC1 by rapamycin induced the formation of septa that separate the nuclei that were formed after mitosis. Regarding neutral lipid metabolism, a higher accumulation of triacylglycerols was not detected, but the cells did contain large lipid bodies, which suggests that small lipid bodies became fused into big lipid droplets. Vacuoles showed a similar behavior as the lipid bodies, and double labeling with Blue-CMAC and BODIPY indicates that vacuoles and lipid bodies were independent organelles. The results suggest that TORC1 has a role in cell morphology, lipid metabolism, and vacuolar physiology in U. maydis.

The endophytic fungus Cladosporium sphaerospermum WBS017 was obtained from healthy bulbs of Fritillaria unibracteata var. wabuensis. Fermentation of C. sphaerospermum on solid rice medium yielded three new hybrid polyketides, cladosins L-N (1-3), and a known derivative cladodionen (4). Further cultivation of this fungus on white bean medium afforded an additional new hybrid polyketide, cladosin O (5) along with three known analogues (6-8). The structures of the new compounds were elucidated using a combination of NMR and HRESIMS data. The absolute configurations of compounds 2 and 3 were determined by Mosher's method and TDDFT-ECD calculations. All isolated compounds were evaluated for their cytotoxic and antimicrobial activities. Cladodionen (4) exhibited cytotoxicity against the mouse lymphoma cell line L5178Y with an IC 50 value of 3.7 μM, and also exhibited antifungal activity against Ustilago maydis and Saccharomyces cerevisiae, while cladosin L (1) displayed week antibacterial activity against Staphylococcus aureus ATCC 29213 and S. aureus ATCC 700699 with MIC values of 50 and 25 μM, respectively., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The emerging resistance of crop pathogens to fungicides poses a challenge to food security and compels discovery of new antifungal compounds. Here, we show that mono-alkyl lipophilic cations (MALCs) inhibit oxidative phosphorylation by affecting NADH oxidation in the plant pathogens Zymoseptoria tritici, Ustilago maydis and Magnaporthe oryzae. One of these MALCs, consisting of a dimethylsulfonium moiety and a long alkyl chain (C 18 -SMe 2 + ), also induces production of reactive oxygen species at the level of respiratory complex I, thus triggering fungal apoptosis. In addition, C 18 -SMe 2 + activates innate plant defense. This multiple activity effectively protects cereals against Septoria tritici blotch and rice blast disease. C 18 -SMe 2 + has low toxicity in Daphnia magna, and is not mutagenic or phytotoxic. Thus, MALCs hold potential as effective and non-toxic crop fungicides.

Divergent and enantiospecific total syntheses of the indolosesquiterpenoids xiamycins A, C, F, H and oridamycin A have been accomplished. The syntheses, which commence from (R)-carvone, employ a key photoinduced benzannulation sequence to forge the carbazole moiety characteristic of these natural products. Late-stage diversification from a common intermediate enabled the first syntheses of xiamycins C and F, and an unexpected one-pot oxidative decarboxylation, which may prove general, led to xiamycin H. All synthetic intermediates and the natural products were tested for anti-fungal activity. Xiamycin H emerged as an inhibitor of three agriculturally relevant fungal pathogens., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: UK-2A is an antifungal antibiotic produced by Streptomyces sp. 517-02. Derivatization of its picolinamide OH to form the isobutyryl acetal led to the discovery of fenpicoxamid (InatreqTM active), which is currently under development as a fungicide by Dow AgroSciences LLC. This paper documents efforts to achieve additional efficacy enhancements through semi-synthetic modification of the benzyl substituent of the UK-2A macrocycle., Results: Of 34 analogs prepared, the most active had mitochondrial electron transport IC 50 values 1.5- to 3.7-fold higher than UK-2A (IC 50 0.86 nM). The cyclohexyl analog (38, IC 50 1.23 nM) was the most intrinsically active derivative, and inhibited in vitro growth of Zymoseptoria tritici (EC 50 2.8 ppb) and Leptosphaeria nodorum (EC 50 6.2 ppb) more strongly than UK-2A (EC 50 5.3 and 11.3 ppb for Z. tritici and L. nodorum, respectively). Heterocyclic ring systems and polar linker functionalities resulted in substantial activity loss. Several analogs (20, 22, 23, 24, 36 and 38) translated Z. tritici in vitro growth inhibition activity to in planta disease control more effectively than did UK-2A, with log D being a key factor in this regard., Conclusions: UK-2A is amenable to further modification at the benzyl position on the macrocycle, which provides opportunities for manipulation of physical properties while retaining strong intrinsic and antifungal activity. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

In the present study we determined whether Ustilago maydis accumulates autophagosomes within vacuoles when the cells are exposed to nutritional stress conditions. We investigated whether proteinase B and proteinase A are involved in their degradation. To this effect, wild type and Δpep4 mutant were incubated in minimal medium lacking a carbon source. It was observed that after incubation in nutrient-deficient media, spherical bodies appeared within the Δpep4 mutant strains vacuoles. In addition, autophagosomes were accumulated in U. maydis WT cells incubated in the presence of the serine protease inhibitor PMSF and accumulation of large autophagosomes and electrodense structures in the Δpep4 mutant cell vacuoles took place. These results demonstrate that the homologues of both, the proteinase B and the protease A, are involved in the autophagosomes degradation process in U. maydis., (© FEMS 2019.)

Background: The antifungal antibiotic UK-2A strongly inhibits mitochondrial electron transport at the Q i site of the cytochrome bc1 complex. Previous reports have described semi-synthetic modifications of UK-2A to explore the structure-activity relationship (SAR), but efforts to replace the picolinic acid moiety have been limited., Results: Nineteen UK-2A analogs were prepared and evaluated for Q i site (cytochrome c reductase) inhibition and antifungal activity. While the majority are weaker Q i site inhibitors than UK-2A (IC 50 , 3.8 nM), compounds 2, 5, 13 and 16 are slightly more active (IC 50 , 3.3, 2.02, 2.89 and 1.55 nM, respectively). Compared to UK-2A, compounds 13 and 16 also inhibit growth of Zymoseptoria tritici and Leptosphaeria nodorum more strongly, while 2 and 13 provide stronger control of Z. tritici and Puccinia triticina in glasshouse tests. The relative activities of compounds 1-19 are rationalized based on a homology model constructed for the Z. tritici Q i binding site. Physical properties of compounds 1-19 influence translation of intrinsic activity to antifungal growth inhibition and in planta disease control., Conclusions: The 3-hydroxy-4-methoxy picolinic acid moiety of UK-2A can be replaced by a variety of o-hydroxy-substituted arylcarboxylic acids that retain strong activity against Z. tritici and other agriculturally relevant fungi. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Enhancing broad-spectrum resistance is a major goal of crop breeding. However, broad-spectrum resistance has not been thoroughly investigated, and its underlying molecular mechanisms remain elusive. In the model plant Arabidopsis ( Arabidopsis thaliana ), ACCELERATED CELL DEATH6 (ACD6) is a key component of broad-spectrum resistance that acts in a positive feedback loop with salicylic acid (SA) to regulate multiple pattern recognition receptors. However, the role of ACD6 in disease resistance in crop plants is unclear. Here, we show that the transcript of ANK23 , one of the 15 ACD6 -like genes in maize ( Zea mays ), is induced by SA and by infection with the pathogenic fungus Ustilago maydis . Heterologous expression of ANK23 restored disease resistance in the Arabidopsis mutant acd6-2 . We show that ANK23 is a maize ortholog of ACD6 and therefore rename ANK23 as ZmACD6 . Furthermore, using CRISPR/Cas9, we generated ZmACD6 knockout maize plants, which are more susceptible to U. maydis than wild-type plants. We also identified a maize line (SC-9) with relatively high ZmACD6 expression levels from a diverse natural maize population. SC-9 has increased disease resistance to U. maydis and defense activation, suggesting a practical approach to cultivate elite varieties with enhanced disease resistance.

Background: The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgr) catalyzes the synthesis of mevalonate, a key compound for the synthesis of cholesterol in humans and ergosterol in fungi. Since the Hmgr enzymes of Saccharomyces cerevisiae, Schizosaccharomyces pombe and Candida glabrata are similar to the Hmgr enzymes of mammals, fungal Hmgr enzymes have been proposed as a model for studying antifungal agents., Aims: To examine the correlation between inhibiting Um-Hmgr enzyme and the viability, sterols synthesis and mating in Ustilago maydis., Methods: Using in silico analysis, the ORF codifying for Um-Hmgr was identified and the protein characteristics were deduced. The effect of the competitive inhibitors of Um-Hmgr on the viability of this basidiomycota, the synthesis of its sterols, and its mating were evaluated., Results: The Umhmgr gene (XP_011389590.1) identified putatively codifies a protein of 1443 aa (ca. MW=145.5kDa) that has a possible binding domain in the endoplasmic reticulum (ER) and high identity with the Hmgr catalytic domain of humans and other yeasts. The inhibition of Um-Hmgr caused a decrease of viability and synthesis of sterols, and also the inhibition of mating. The activity of Um-Hmgr is mainly located in the membrane fraction of the fungus., Conclusions: Given our results we believe U. maydis is a valid model for studying synthetic inhibitors with lipid-lowering or antifungal activity. Additionally, we propose the Hmgr enzyme as an alternative molecular target to develop compounds for treating both phytopathogenic and pathogenic human fungi., (Copyright © 2019 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.)

DNA-protein cross-links (DPCs) are frequently occurring lesions that provoke continual threats to the integrity of the genome by interference with replication and transcription. Reactive aldehydes generated from endogenous metabolic processes or produced in the environment are sources that trigger cross-linking of DNA with associated proteins. DNA repair pathways in place for removing DPCs, or for bypassing them to enable completion of replication, include homologous recombination (HR) and replication fork remodeling (FR) systems. Here, we surveyed a set of mutants defective in known HR and FR components to determine their contribution toward maintaining resistance to chronic formaldehyde (FA) exposure in Ustilago maydis , a fungus that relies on the BRCA2-family member Brh2 as the principal Rad51 mediator in repair of DNA strand breaks. We found that, in addition to Brh2, Rad52 was also vital for resistance to FA. Deleting the gene for Rec8, a kleisin subunit of cohesin, eliminated the requirement for Brh2, but not Rad52, in FA resistance. The Rad51 K133R mutant variant that is able to bind DNA but unable to dissociate from it was able to support resistance to FA. These findings suggest a model for DPC repair and tolerance that features a specialized role for Rad52, enabling Rad51 to access DNA in its noncanonical capacity of replication fork protection rather than DNA strand transfer., (Copyright © 2018 by the Genetics Society of America.)

Cells maintain a small arsenal of resolving functions to process and eliminate complex DNA intermediates that result as a consequence of homologous recombination and distressed replication. Ordinarily the homologous recombination system serves as a high-fidelity mechanism to restore the integrity of a damaged genome, but in the absence of the appropriate resolving function it can turn DNA intermediates resulting from replication stress into pathological forms that are toxic to cells. Here we have investigated how the nucleases Mus81 and Gen1 and the helicase Blm contribute to survival after DNA damage or replication stress in Ustilago maydis cells with crippled yet homologous recombination-proficient forms of Brh2, the BRCA2 ortholog and primary Rad51 mediator. We found collaboration among the factors. Notable were three findings. First, the ability of Gen1 to rescue hydroxyurea sensitivity of dysfunctional Blm requires the absence of Mus81. Second, the response of mutants defective in Blm and Gen1 to hydroxyurea challenge is markedly similar suggesting cooperation of these factors in the same pathway. Third, the repair proficiency of Brh2 mutant variants deleted of its N-terminal DNA binding region requires not only Rad52 but also Gen1 and Mus81. We suggest these factors comprise a subpathway for channeling repair when Brh2 is compromised in its interplay with DNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The fungal pathogen Ustilago maydis causes disease on maize by mating to establish an infectious filamentous cell type that invades the host and induces tumours. We previously found that β-oxidation mutants were defective in virulence and did not grow on acetate. Here, we demonstrate that acetate inhibits filamentation during mating and in response to oleic acid. We therefore examined the influence of different carbon sources by comparing the transcriptomes of cells grown on acetate, oleic acid or glucose, with expression changes for the fungus during tumour formation in planta. Guided by the transcriptional profiling, we found that acetate negatively influenced resistance to stress, promoted the formation of reactive oxygen species, triggered cell death in stationary phase and impaired virulence on maize. We also found that acetate induced mitochondrial stress by interfering with mitochondrial functions. Notably, the disruption of oxygen perception or inhibition of the electron transport chain also influenced filamentation and mating. Finally, we made use of the connections between acetate and β-oxidation to test metabolic inhibitors for an influence on growth and virulence. These experiments identified diclofenac as a potential inhibitor of virulence. Overall, these findings support the possibility of targeting mitochondrial metabolic functions to control fungal pathogens., (© 2017 John Wiley & Sons Ltd.)

Chitosan is a stressing molecule that affects the cells walls and plasma membrane of fungi. For chitosan derivatives, the action mode is not clear. In this work, we used the yeast Ustilago maydis to study the effects of these molecules on the plasma membrane, focusing on physiologic and stress responses to chitosan (CH), oligochitosan (OCH), and glycol-chitosan (GCH). Yeasts were cultured with each of these molecules at 1 mg·mL -1 in minimal medium. To compare plasma membrane damage, cells were cultivated in isosmolar medium. Membrane potential (Δψ) as well as oxidative stress were measured. Changes in the total plasma membrane phospholipid and protein profiles were analyzed using standard methods, and fluorescence-stained mitochondria were observed. High osmolarity did not protect against CH inhibition and neither affected membrane potential. The OCH did produce higher oxidative stress. The effects of these molecules were evidenced by modifications in the plasma membrane protein profile. Also, mitochondrial damage was evident for CH and OCH, while GCH resulted in thicker cells with fewer mitochondria and higher glycogen accumulation., Competing Interests: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Regulation of genes involved in nitrogen metabolism likely plays a role in the ability of fungi to exploit and survive under different environmental situations. To learn about the mechanism of adaptation of the biotrophic fungus Ustilago maydis from a medium containing a source of fixed nitrogen, to a medium depending on the ability to fix N 2 by its bacterial endosymbiont, we explored gene expression profiles using RNA-Seq analyses under these two conditions. The differentially expressed (DE) fungal genes were analyzed, identifying 90 genes that were regulated 24 h after shifting the fungus to media lacking ammonium nitrate as a nitrogen source. From these, mRNA levels were increased for 49 genes, whereas 41 were down-regulated. The functional description associated to the regulated genes revealed that nine key pathways were represented, including, secondary metabolism, the metabolism of nitrogen, amino acid, fatty acid, amino sugar and nucleotide sugar, purine, peroxisome, and the regulation of actin cytoskeleton. These results suggest that the interplay of U. maydis with its N 2 fixing bacterial endosymbiont is a flexible process that may be active during the adaptation of the fungus to the different nitrogen sources., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Hybrid histidine kinase is part of a two-component system that is required for various stress responses and pathogenesis of pathogenic fungi. The Tco1 gene in human pathogen Cryptococcus neoformans encodes a hybrid histidine kinase and is important for pathogenesis. In this study, we identified a Tco1 homolog, UmTco1 , in the maize pathogen Ustilago maydis by bioinformatics analysis. To explore the role of UmTco1 in the survival of U. maydis under environmental stresses and its pathogenesis, Δumtco1 mutants were constructed by allelic exchange. The growth of Δumtco1 mutants was significantly impaired when they were cultured under hyperosmotic stress. The Δumtco1 mutants exhibited increased resistance to antifungal agent fludioxonil. In particular, the Δumtco1 mutants were unable to produce cytokinesis or conjugation tubes, and to develop fuzzy filaments, resulting in impaired mating between compatible strains. The expression levels of Prf1, Pra1 , and Mfa1 , which are involved in the pheromone pathway, were significantly decreased in the Δumtco1 mutants. In inoculation tests to the host plant, the Δumtco1 mutants showed significantly reduced ability in the production of anthocyanin pigments and tumor development on maize leaves. Overall, the combined results indicated that UmTco1 plays important roles in the survival under hyperosmotic stress, and contributes to cytokinesis, sexual development, and virulence of U. maydis by regulating the expression of the genes involved in the pheromone pathway.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective/background: Microbial infections such as tuberculosis is a major cause of mortality worldwide. Plant-derived phytochemicals have a long history of providing much-needed novel therapeutics. Triterpenoids are among the prominent phytochemicals that possess numerous biological activities. Among them is maslinic acid (MA), a biologically active olean-type pentacyclic triterpenoid. In search of a novel antimicrobial agent, we aimed to evaluate the antimicrobial potential of MA., Methods: Antibacterial and antifungal activity was evaluated through the agar well diffusion method. Antitubercular activity was analysed through the agar well diffusion and disc diffusion methods, respectively. Antioxidant capacity was determined through assays for total antioxidant capacity, 2,2-diphenyl-1-picrylhydrazyl radical scavenging, hydrogen peroxide radical scavenging, and Fe 3+ reducing power. The program Prediction of Activity Spectra for Substances was used to calculate the possible biological activity of MA., Results: MA showed dose-dependent antioxidant activity similar to that of ascorbic acid. It had no inhibitory effect on bacterial strains, but it had moderate activity against the fungi Aspergillus flavus and Ustilago maydis, with Aspergillus niger being the most sensitive to MA. MA also exhibited strong antimycobacterial activity. Probable antioxidant, antibacterial, and antifungal activity of MA based on software calculations are 0.479, 0.363 and 0.589 respectively., Conclusion: This work provides scientific evidence of the antioxidant, antifungal, and antimycobacterial activities of MA, showing its potential application in the development of natural antioxidants and antimicrobial agents for the agro-food and pharmaceutical industries., (Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.)

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.

Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*)rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1(Q488*) mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ustilago maydis, a dimorphic fungus causing corn smut disease, serves as an excellent model to study different aspects of cell development. This study shows the influence of chitosan, oligochitosan and glycol chitosan on cell growth and physiology of U. maydis. These biological macromolecules affected the cell growth of U. maydis. In particular, it was found that chitosan completely inhibited U. maydis growth at 1mg/mL concentration. Microscopic studies revealed swellings on the surface of the cells treated with the polymers, and chitosan caused complete destruction of the membrane and formation of vesicles on the periphery of the cell. Oligochitosan and chitosan caused changes in oxygen consumption, K(+) efflux and H(+)-ATPase activity. Oligochitosan induced a faster consumption of oxygen in the cells, while glycol chitosan provoked slower oxygen consumption. It is noteworthy that chitosan completely inhibited the fungal respiratory activity. The strongest effects were exhibited by chitosan in all evaluated aspects. These findings showed high sensitivity of U. maydis to chitosan and provided evidence for antifungal effects of chitosan derivatives. To our knowledge, this is a first report showing that chitosan and its derivatives affect the cell morphology and physiological processes in U. maydis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We observed that the maize pathogenic fungus Ustilago maydis grew in nitrogen (N)-free media at a rate similar to that observed in media containing ammonium nitrate, suggesting that it was able to fix atmospheric N2 . Because only prokaryotic organisms have the capacity to reduce N2 , we entertained the possibility that U. maydis was associated with an intracellular bacterium. The presence of nitrogenase in the fungus was analyzed by acetylene reduction, and capacity to fix N2 by use of (15) N2 . Presence of an intracellular N2 -fixing bacterium was analyzed by PCR amplification of bacterial 16S rRNA and nifH genes, and by microscopic observations. Nitrogenase activity and (15) N incorporation into the cells proved that U. maydis fixed N2 . Light and electron microscopy, and fluorescence in situ hybridization (FISH) experiments revealed the presence of intracellular bacteria related to Bacillus pumilus, as evidenced by sequencing of the PCR-amplified fragments. These observations reveal for the first time the existence of an endosymbiotic N2 -fixing association involving a fungus and a bacterium., (© 2015 The Authors New Phytologist © 2015 New Phytologist Trust.)

Ustilago esculenta grows within the flowering stem of the aquatic grass Zizania latifolia, resembling a fungal endophyte. The fungus colonizes Z. latifolia and induces swelling which results in the formation of galls near the base of the plant. Due to their unique flavor and textures these galls are considered as a delicacy in southern China. Efficient genetic manipulation is required to determine the relationship between U. esculenta and Z. latifolia. In this study, we report a protoplast-based transformation system for this unique fungal species. We have explored various factors (enzyme digesting conditions, osmotic pressure stabilizers, vectors and selection agents) that might impact protoplast yield and high frequencies of transformation. A haploid strain (UeT55) of U. esculenta was found to produce higher yields of protoplasts when treating with 15 mg mL(-1) lywallzyme in a sucrose-containing solution at 30°C for 3 h. The transformation frequencies were higher when fungal strain was transformed with a linear plasmid harboring hygromycin or carboxin resistance gene and regenerated on a sucrose-containing medium. A UeICL gene (coding isocitrate lyase) was disrupted and an EGFP (coding enhanced green fluorescent protein) gene was overexpressed successfully in the UeT55 strain using the developed conditions. The genetic manipulation system reported in this study will open up new opportunities for forward and reverse genetics in U. esculenta., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Alkaline pH triggers an adaptation mechanism in fungi that is mediated by Rim101/PacCp, a zinc finger transcription factor. To identify the genes under its control in Ustilago maydis, we performed microarray analyses, comparing gene expression in a wild-type strain versus a rim101/pacC mutation strain of the fungus. In this study we obtained evidence of the large number of genes regulated mostly directly, but also indirectly (probably through regulation of other transcription factors), by Rim101/PacCp, including proteins involved in a large number of physiological activities of the fungus. Our analyses suggest that the response to alkaline conditions under the control of the Pal/Rim pathway involves changes in the cell wall and plasma membrane through alterations in their lipid, protein and polysaccharide composition, changes in cell polarity, actin cytoskeleton organization, and budding patterns. Also as expected, adaptation involves regulation by Rim101/PacC of genes involved in meiotic functions, such as recombination and segregation, and expression of genes involved in ion and nutrient transport, as well as general vacuole functions., (© 2014 The Authors.)

Early endosomes (EEs) mediate protein sorting, and their cytoskeleton-dependent motility supports long-distance signaling in neurons. Here, we report an unexpected role of EE motility in distributing the translation machinery in a fungal model system. We visualize ribosomal subunit proteins and show that the large subunits diffused slowly throughout the cytoplasm (Dc,60S = 0.311 µm(2)/s), whereas entire polysomes underwent long-range motility along microtubules. This movement was mediated by "hitchhiking" on kinesin-3 and dynein-driven EEs, where the polysomes appeared to translate EE-associated mRNA into proteins. Modeling indicates that this motor-driven transport is required for even cellular distribution of newly formed ribosomes. Indeed, impaired EE motility in motor mutants, or their inability to bind EEs in mutants lacking the RNA-binding protein Rrm4, reduced ribosome transport and induced ribosome aggregation near the nucleus. As a consequence, cell growth was severely restricted. Collectively, our results indicate that polysomes associate with moving EEs and that "off- and reloading" distributes the protein translation machinery.

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., (© 2012 Society of Chemical Industry.)

Ustilago maydis (DC) Cda., a phytopathogenic Basidiomycota, is the causal agent of corn smut. During its life cycle U. maydis alternates between a yeast-like, haploid nonpathogenic stage, and a filamentous, dikaryotic pathogenic form that invades the plant and induces tumor formation. As all the members of the Subphylum Ustilaginomycotina, U. maydis is unable to form basidiocarps, instead it produces teliospores within the tumors that germinate forming a septate basidium (phragmobasidium). We have now established conditions allowing a completely different developmental program of U. maydis when grown on solid medium containing auxins in dual cultures with maize embryogenic calli. Under these conditions U. maydis forms large hemi-spheroidal structures with all the morphological and structural characteristics of gastroid-type basidiocarps. These basidiocarps are made of three distinct hyphal layers, the most internal of which (hymenium) contains non-septate basidia (holobasidia) from which four basidiospores develop. In basidiocarps meiosis and genetic recombination occur, and meiotic products (basidiospores) segregate in a Mendelian fashion. These results are evidence of sexual cycle completion of an Ustilaginomycotina in vitro, and the demonstration that, besides its quasi-obligate biotrophic pathogenic mode of life, U. maydis possesses the genetic program to form basidiocarps as occurs in saprophytic Basidiomycota species., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Protein kinases are key enzymes in the complex regulation of cellular processes in almost all living organisms. For this reason, protein kinases represent attractive targets to stop the growth of eukaryotic pathogens such as protozoa and fungi. However, using kinase inhibitors to fight against these organisms bears several challenges since most of them are unselective and will also affect crucial host kinases. Here we present the X-ray structure of glycogen synthase kinase 3 from the fungal plant pathogen Ustilago maydis (UmGSK3) and its inhibition by type-II kinase inhibitors. Despite the high sequence homology between the human and the fungal variant of this vital kinase, we found substantial differences in the conformational plasticity of their active sites. Compounds that induced such conformational changes could be used to selectively inhibit the fungal kinase. This study serves as an example of how species-specific selectivity of inhibitors can be achieved by identifying and addressing the inactive state of a protein kinase. In addition to this, our study gives interesting insights into the molecular plasticity of UmGSK3 by revealing a previously unknown inactive conformation of this important kinase family.

The Transfersome® is a lipid vesicle that contains membrane softeners, such as Tween 80, to make it ultra-deformable. This feature makes the Transfersome® an efficient carrier for delivery of therapeutic drugs across the skin barrier. It was reported that TDT 067 (a topical formulation of 15 mg/ml terbinafine in Transfersome® vesicles) has a much more potent antifungal activity in vitro compared with conventional terbinafine, which is a water-insoluble fungicide. Here we use ultra-structural studies and live imaging in a model fungus to describe the underlying mode of action. We show that terbinafine causes local collapse of the fungal endoplasmic reticulum, which was more efficient when terbinafine was delivered in Transfersome® vesicles (TFVs). When applied in liquid culture, fluorescently labeled TFVs rapidly entered the fungal cells (T(1/2)~2 min). Entry was F-actin- and ATP-independent, indicating that it is a passive process. Ultra-structural studies showed that passage through the cell wall involves significant deformation of the vesicles, and depends on a high concentration of the surfactant Tween 80 in their membrane. Surprisingly, the TFVs collapsed into lipid droplets after entry into the cell and the terbinafine was released from their interior. With time, the lipid bodies were metabolized in an ATP-dependent fashion, suggesting that cytosolic lipases attack and degrade intruding TFVs. Indeed, the specific monoacylglycerol lipase inhibitor URB602 prevented Transfersome® degradation and neutralized the cytotoxic effect of Transfersome®-delivered terbinafine. These data suggest that (a) Transfersomes deliver the lipophilic fungicide Terbinafine to the fungal cell wall, (b) the membrane softener Tween 80 allows the passage of the Transfersomes into the fungal cell, and (c) fungal lipases digest the invading Transfersome® vesicles thereby releasing their cytotoxic content. As this mode of action of Transfersomes is independent of the drug cargo, these results demonstrate the potential of Transfersomes in the treatment of all fungal diseases.

Infestation of crops by pathogenic fungi has continued to have a major impact by reducing yield and quality, emphasizing the need to identify new targets and develop new agents to improve methods of crop protection. Here we present Aurora kinase from the phytopathogenic fungus Ustilago maydis as a novel target for N-substituted diaminopyrimidines, a class of small-molecule kinase inhibitors. We show that Aurora kinase is essential in U. maydis and that diaminopyrimidines inhibit its activity in vitro. Furthermore, we observed an overall good correlation between in vitro inhibition of Aurora kinase and growth inhibition of diverse fungi in vivo. In vitro inhibition assays with Ustilago and human Aurora kinases indicate that some compounds of the N-substituted diaminopyrimidine class show specificity for the Ustilago enzyme, thus revealing their potential as selective fungicides.

Synthesis of some new substituted [3-(3-chloro-4-fluorophenyl)-2-oxo-3,4-dihydro-2H-2lambda(5)-benzo[e][1,3,2]oxazaphosphinin-2-yl]-(aryl/alkyl)methanols (7a-k) based on the Pudovick reaction was accomplished in the presence of niobium pentoxide (Nb(2)O(5)) without using an external chiral ligand. Nb(2)O(5) appears to form the metal complex intermediate catalyst system (6) by reacting with 3-(3-chloro-4-fluoro-phenyl)-3,4-dihydrobenzo[e][1,3,2]oxazaphosphinine-2-oxide (4), which not only directs the Pudovick addition reactions of aldehyde but also increases the yields and purity of the products. These compounds exhibited a lethal effect on whip smut of sugarcane and were degraded in the environment in the presence of bacteria and fungi to nontoxic phosphate residues that act as possible plant nutrients. Thus, a new class of benzooxazaphosphininyl methanol derivatives that act in synergy both as antipathogens and as plant nutrients in the environment have been discovered.

The fungus Ustilago maydis is a biotrophic pathogen of maize (Zea mays). In its genome we have identified an ortholog of YAP1 (for Yeast AP-1-like) from Saccharomyces cerevisae that regulates the oxidative stress response in this organism. yap1 mutants of U. maydis displayed higher sensitivity to H(2)O(2) than wild-type cells, and their virulence was significantly reduced. U. maydis yap1 could partially complement the H(2)O(2) sensitivity of a yap1 deletion mutant of S. cerevisiae, and a Yap1-green fluorescent protein fusion protein showed nuclear localization after H(2)O(2) treatment, suggesting that Yap1 in U. maydis functions as a redox sensor. Mutations in two Cys residues prevented accumulation in the nucleus, and the respective mutant strains showed the same virulence phenotype as Deltayap1 mutants. Diamino benzidine staining revealed an accumulation of H(2)O(2) around yap1 mutant hyphae, which was absent in the wild type. Inhibition of the plant NADPH oxidase prevented this accumulation and restored virulence. During the infection, Yap1 showed nuclear localization after penetration up to 2 to 3 d after infection. Through array analysis, a large set of Yap1-regulated genes were identified and these included two peroxidase genes. Deletion mutants of these genes were attenuated in virulence. These results suggest that U. maydis is using its Yap1-controlled H(2)O(2) detoxification system for coping with early plant defense responses.

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.

Fungal chitin synthases (CHSs) form fibers of the cell wall and are crucial for substrate invasion and pathogenicity. Filamentous fungi contain up to 10 CHSs, which might reflect redundant functions or the complex biology of these fungi. Here, we investigate the complete repertoire of eight CHSs in the dimorphic plant pathogen Ustilago maydis. We demonstrate that all CHSs are expressed in yeast cells and hyphae. Green fluorescent protein (GFP) fusions to all CHSs localize to septa, whereas Chs5-GFP, Chs6-GFP, Chs7-yellow fluorescent protein (YFP), and Myosin chitin synthase1 (Mcs1)-YFP were found at growth regions of yeast-like cells and hyphae, indicating that they participate in tip growth. However, only the class IV CHS genes chs7 and chs5 are crucial for shaping yeast cells and hyphae ex planta. Although most CHS mutants were attenuated in plant pathogenicity, Deltachs6, Deltachs7, and Deltamcs1 mutants were drastically reduced in virulence. Deltamcs1 showed no morphological defects in hyphae, but Mcs1 became essential during invasion of the plant epidermis. Deltamcs1 hyphae entered the plant but immediately lost growth polarity and formed large aggregates of spherical cells. Our data show that the polar class IV CHSs are essential for morphogenesis ex planta, whereas the class V myosin-CHS is essential during plant infection.

Saccharum officinarum, cv. Mayarí, is a variety of sugarcane resistant to smut disease caused by Ustilago scitaminea. Sugarcane naturally produces glycoproteins that accumulate in the parenchymatous cells of stalks. These glycoproteins contain a heterofructan as polysaccharide moiety. The concentration of these glycoproteins clearly increases after inoculation of sugarcane plants with smut teliospores, although major symptoms of disease are not observed. These glycoproteins induce homotypic adhesion and inhibit teliospore germination. When glycoproteins from healthy, non-inoculated plants are fractionated, they inhibit actin capping, which occurs before teliospore germination. However, inoculation of smut teliospores induce glycoprotein fractions that promote teliospore polarity and are different from those obtained from healthy plants. These fractions exhibit arginase activity, which is strongly enhanced in inoculated plants. Arginase from healthy plants binds to cell wall teliospores and it is completely desorpted by sucrose, but only 50% of arginase activity from inoculated plants is desorpted by the disaccharide. The data presented herein are consistent with a model of excess arginase entry into teliospores. Arginase synthesized by sugarcane plants as a response to the experimental infection would increase the synthesis of putrescine, which impedes polarization at concentration values higher than 0.05 mM. However, smut teliospores seem to be able to change the pattern of glycoprotein production by sugarcane, thereby promoting the synthesis of different glycoproteins that activate polarization after binding to their cell wall ligand.

Mutants of Ustilago maydis (DC) Corda with high resistance to azoxystrobin (RF 164 to 4714, based on EC50 values), an inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated in a mutation frequency of 2.3 x 10(-7) after nitrosoguanidine mutagenesis and selection on media containing 1 microgram ml-1 azoxystrobin in addition to 0.5 mM salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strains by azoxystrobin (1.5 micrograms ml-1) in addition to SHAM (1 mM), but not in the mutant isolates. Genetic analysis with nine such mutant isolates resulted in progeny phenotypes which did not follow Mendelian segregation, but satisfied the criteria of non-Mendelian (cytoplasmic) heredity. In crosses between three mutant isolates with the compatible wild-type strains, the sensitivity was inherited by progeny maternally from the wild-type parent strain (criterion of uniparental inheritance). In crosses between wild-type strains and remaining mutant isolates, a continuous distribution of sensitivity in the progeny was found (criterion of vegetative segregation). The third criterion of cytoplasmic resistance (criterion of intracellular selection) was fulfilled by experiments on the stability of resistance phenotypes. With two exceptions, a reduction of resistance was observed in the mutant strains when they were grown on inhibitor-free medium. Recovery of the high resistance level was observed after they were returned to the selection medium. Cross-resistance studies with other fungicides, which also inhibit electron transport through complex III of respiratory chain, showed that mutations for resistance to azoxystrobin were also responsible for reduced sensitivity to kresoxim-methyl (RF 18 to 1199) and to antimycin-A (RF 20 to 305), which act at the Qo and Qi sites of the cytochrome bc1 complex, respectively. Studies of the fitness of azoxystrobin-resistant isolates showed that these mutations appeared to be pleiotropic, having significant adverse effects on growth in liquid culture and pathogenicity on young corn plants.

Aigialomycins A-E (2-6), new 14-membered resorcylic macrolides, were isolated together with a known hypothemycin (1) from the mangrove fungus, Aigialus parvus BCC 5311. Structures of these compounds, including absolute configuration, were elucidated by spectroscopic methods, chemical conversions, and X-ray crystallographic analysis. Hypothemycin and aigialomycin D (5) exhibited in vitro antimalarial activity with IC(50) values of 2.2 and 6.6 microg/mL, respectively, while other analogues were inactive. Cytotoxicities of these compounds were also evaluated.

The endoplasmic reticulum (ER) of most vertebrate cells is spread out by kinesin-dependent transport along microtubules, whereas studies in Saccharomyces cerevisiae indicated that motility of fungal ER is an actin-based process. However, microtubules are of minor importance for organelle transport in yeast, but they are crucial for intracellular transport within numerous other fungi. Herein, we set out to elucidate the role of the tubulin cytoskeleton in ER organization and dynamics in the fungal pathogen Ustilago maydis. An ER-resident green fluorescent protein (GFP)-fusion protein localized to a peripheral network and the nuclear envelope. Tubules and patches within the network exhibited rapid dynein-driven motion along microtubules, whereas conventional kinesin did not participate in ER motility. Cortical ER organization was independent of microtubules or F-actin, but reformation of the network after experimental disruption was mediated by microtubules and dynein. In addition, a polar gradient of motile ER-GFP stained dots was detected that accumulated around the apical Golgi apparatus. Both the gradient and the Golgi apparatus were sensitive to brefeldin A or benomyl treatment, suggesting that the gradient represents microtubule-dependent vesicle trafficking between ER and Golgi. Our results demonstrate a role of cytoplasmic dynein and microtubules in motility, but not peripheral localization of the ER in U. maydis.

A series of novel (hetero) aryloxylepidine derivatives was devised as hybrid structures of the phenoxyquinoline and phenethoxyquin(az)oline fungicides. Synthesis of these targets required the development of several new routes to derivatised 4-hydroxymethylquinolines, and subsequent coupling with phenols or haloarenes. The aryloxylepidines generally showed moderate broad-spectrum fungicidal activity across several diseases of cereals. Substitution of the quinoline ring with chlorine at the 7- and/or 5-positions gave molecules with high levels of protectant activity against Erysiphe graminis f sp tritici (powdery mildew of wheat), but this did not improve the level of fungicidal activity against other diseases. In vitro activity against mitochondrial electron transport complex I (MET) derived from Ustilago maydis showed that 8-fluorolepidine analogues were moderately active at this target site, while the more fungicidally active 7- and 5,7-substituted compounds were inactive. This indicates that MET is not the primary target of these highly active powdery mildewicides.

KP4 is a virally encoded fungal toxin secreted by the P4 killer strain of Ustilago maydis. From our previous structural studies, it seemed unlikely that KP4 acts by forming channels in the target cell membrane. Instead, KP4 was proposed to act by blocking fungal calcium channels, as KP4 was shown to inhibit voltage-gated calcium channels in rat neuronal cells, and its effects on fungal cells were abrogated by exogenously added calcium. Here, we extend these studies and demonstrate that KP4 acts in a reversible manner on the cell membrane and does not kill the cells, but rather inhibits cell division. This action is mimicked by EGTA and is abrogated specifically by low concentrations of calcium or non-specifically by high ionic strength buffers. We also demonstrate that KP4 affects (45)Ca uptake in U. maydis. Finally, we show that cAMP and a cAMP analogue, N 6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, both abrogate KP4 effects. These results suggest that KP4 may inhibit cell growth and division by blocking calcium-regulated signal transduction pathways.

The REC2 gene of Ustilago maydis encodes a homologue of the Escherichia coli RecA protein and was first identified in a screen for UV-sensitive mutants. The original isolate, rec2-1, was found to be deficient in repair of DNA damage, genetic recombination and meiosis. We report here that the rec2-197 allele, which was constructed by gene disruption, retains some biological activity and is partially dominant with respect to REC2. The basis for the residual activity is probably as a result of expression of a diffusible product from the rec2-197 allele that augments or interferes with REC2 functions. This product appears to be a polypeptide expressed from a remnant of the 5' end of the open reading frame that was not removed in creating the gene disruption. The mutator activity and disturbed meiosis of rec2-197 suggest that the Rec2 protein functions in a process that avoids spontaneous mutation and insures faithful meiotic chromosome segregation. A prediction based on the phenotype of rec2-197 is that Rec2 protein interacts with one or more other proteins in directing these functions. To identify interacting proteins we performed a yeast two-hybrid screen and found Rad51 as a candidate. Rec2-197 and Rad51 appear to interact to a similar degree.

The cAMP signal transduction pathway mediates the switch between yeast-like and filamentous growth and influences both sexual development and pathogenicity in the smut fungus Ustilago maydis. Signaling via cAMP may also play a role in fungicide resistance in U. maydis. In particular, the adr1 gene, which encodes the catalytic subunit of the U. maydis cAMP-dependent protein kinase (PKA), is implicated in resistance to the dicarboximide and aromatic hydrocarbon fungicides. In this study, we examined the sensitivity of PKA to vinclozolin and could not demonstrate direct inhibition of protein kinase activity. However, we did find that mutants with disruptions in the ubc1 gene, which encodes the regulatory subunit of PKA, were resistant to both vinclozolin and chloroneb. We also found that these fungicides altered the morphology of both wild-type and ubc1 mutant cells. In addition, strains that are defective in ubc1 display osmotic sensitivity, a property often associated with vinclozolin and chloroneb resistance in other fungi., (Copyright 2001 Academic Press.)

Ustilago maydis is an edible parasitic basidiomycete, which specifically infects corn (Zea mays) and teocintle (Z. diploperennis). To characterise the interaction between the basidiomycete and its host organism, we tested the effect of plant lectins with well-known sugar specificity on the growth and germination of U. maydis spores. Lectins specific for N-acetyl-D-galactosamine, such as those from Dolichos biflorus and Phaseolus lunatus, and the wheatgerm agglutinin specific for N-acetyl-D-glucosamine inhibited spore germination, but were ineffective in modifying U. maydis cell growth. The galactose-specific lectin from the corn coleoptyle inhibited both germination and cell growth, while the lectin concanavalin A (mannose/glucose specific) activated spore germination and growth. Our results suggest that specific saccharide-containing receptors participate in regulating the growth and maturation of U. maydis spores.

The cDNA encoding the antifungal protein KP4 from Ustilago maydis-infecting virus was inserted behind the ubiquitin promoter of maize and genetically transferred to wheat varieties particularly susceptible to stinking smut (Tilletia tritici) disease. The transgene was integrated and inherited over several generations. Of seven transgenic lines, three showed antifungal activity against U. maydis. The antifungal activity correlated with the presence of the KP4 transgene. KP4-transgenic, soil-grown wheat plants exhibit increased endogenous resistance against stinking smut.

Shuttle vectors with new or improved features were constructed to enable facile genetic manipulations in the plant pathogen Ustilago maydis. Sets of plasmids selectable in media containing geneticin, carboxin, nourseothricin, or hygromycin, able to replicate autonomously, to transform U. maydis by integration, and to express foreign genes under control of the homologous glyceraldehyde-3-phosphate dehydrogenase promoter, were built upon a common pUC19 vector backbone. This permits a large number of choices for a cloning site, blue/white screening for recombinant plasmids, rapid transfer of a cloned DNA fragment between plasmids, and choice of several dominant drug-resistance markers for selection in U. maydis.

Cell-cell signaling is an integral part of the sexual and disease cycles of the smut fungi, which must mate to be pathogenic. This study reports the cloning and characterization of the pheromone genes Uhmfa1 and Uhmfa2 from MAT-1 and MAT-2 mating types of U. hordei, respectively, and the pheromone receptor gene Uhpra2 from MAT-2 cells. Similar to other fungal pheromone genes, Uhmfa1 and Uhmfa2 encode precursor peptides. Uhpra2 encodes a protein with sequence similarity to the 7-transmembrane class of G-protein coupled receptors. Deletion of Uhmfa1 and Uhpra1, and their subsequent replacement, confirmed the role of these genes in initiation of the sexual cycle. Uhmfa1 and Uhmfa2 were differentially expressed in various cell types and when opposite mating-type cells were grown together. The predicted mature pheromones of each mating type were synthesized, and each specifically induced conjugation tube formation in cells of the opposite mating type.

Modulation of the expression of the Ustilago maydis Pyr3 gene, encoding dihydroorotase (DHOase), through antisense RNA regulation has been explored. This was done by placing the gene in sense and antisense orientations under the control of an hsp70-like gene promoter in a high-copy number autonomously replicating expression vector. Cells transformed with the antisense vector contained similar levels of DHOase activity to those found in cells harboring the expression vector alone. Transformants containing the antisense vector did not exhibit uridine-dependent growth, which would be expected for a Pyr3-deficient phenocopy. This was despite detection of high levels of antisense RNA transcripts in cells transformed with the Pyr3 antisense vector.

In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein alpha subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition, we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U. maydis and discuss how this pathway feeds into the pheromone response.

In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells.