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Ustilago maydis and Ustilago cynodontis are natural producers of a broad range of valuable molecules including itaconate, malate, glycolipids, and triacylglycerols. Both Ustilago species are insensitive toward medium impurities, and have previously been engineered for efficient itaconate production and stabilized yeast-like growth. Due to these features, these strains were already successfully used for the production of itaconate from different alternative feedstocks such as molasses, thick juice, and crude glycerol. Here, we analyzed the amylolytic capabilities of Ustilago species for metabolization of starch, a highly abundant and low-cost polymeric carbohydrate widely utilized as a substrate in several biotechnological processes. Ustilago cynodontis was found to utilize gelatinized potato starch for both growth and itaconate production, confirming the presence of extracellular amylolytic enzymes in Ustilago species. Starch was rapidly degraded by U. cynodontis, even though no α-amylase was detected. Further experiments indicate that starch hydrolysis is caused by the synergistic action of glucoamylase and α-glucosidase enzymes. The enzymes showed a maximum activity of around 0.5 U ml-1 at the fifth day after inoculation, and also released glucose from additional substrates, highlighting potential broader applications. In contrast to U. cynodontis, U. maydis showed no growth on starch accompanied with no detectable amylolytic activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Fungal pathogenesis depends on accurate secretion and location of virulence factors which drive host colonization. Protein glycosylation is a common posttranslational modification of cell wall components and other secreted factors, typically required for correct protein localization, secretion and function. Thus, the absence of glycosylation is associated with animal and plant pathogen avirulence. While the relevance of protein glycosylation for pathogenesis has been well established, the main glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Here, we devise a proteomics approach to identify such proteins and use it to demonstrate a role for the highly conserved protein disulfide isomerase Pdi1 in virulence. We show that efficient Pdi1 N-glycosylation, which promotes folding into the correct protein conformation, is required for full pathogenic development of the corn smut fungus Ustilago maydis. Remarkably, the observed virulence defects are reminiscent of those seen in glycosylation-defective cells suggesting that the N-glycosylation of Pdi1 is necessary for the full secretion of virulence factors. All these observations, together with the fact that Pdi1 protein and RNA expression levels rise upon virulence program induction, suggest that Pdi1 glycosylation is important for normal pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis., Competing Interests: The authors have declared that no competing interests exist.

Covering: up to the end of 2018Soraphens are a class of polyketide natural products discovered from the myxobacterial strain Sorangium cellulosum. The review is intended to provide an overview on the biosynthesis, chemistry and biological properties of soraphens, that represent a prime example to showcase the value of natural products as tools to decipher cell biology, but also to open novel therapeutic options. The prototype soraphen A is an inhibitor of acetyl coenzyme A carboxylase (ACC1/2), an enzyme that converts acetyl-CoA to malonyl-CoA and thereby controls essential cellular metabolic processes like lipogenesis and fatty acid oxidation. Soraphens illustrate how the inhibition of a single target (ACC1/2) may be explored to treat various pathological conditions: initially developed as a fungicide, efforts in the past decade were directed towards human diseases, including diabetes/obesity, cancer, hepatitis C, HIV, and autoimmune disease - and led to a synthetic molecule, discovered by virtual screening of the allosteric binding site of soraphen in ACC, that is currently in phase 2 clinical trials. We will summarize how structural analogs of soraphen A have been generated through extensive isolation efforts, genetic engineering of the biosynthetic gene cluster, semisynthesis as well as partial and total synthesis.

Background: Ustilago esculenta, a typical dimorphic fungus could infect Zizania latifolia and induce host stem swollen to form an edible vegetable called Jiaobai in China. The strains differentiation especially in the mating ability and pathogenicity is closely related to different phenotypes of Jiaobai formed in the fields. Dimorphic switching, a tightly regulated processes, is essential for the pathogenetic development of dimorphic fungi. In responses to environment cues, dimorphic switching can be activated through two conserved cell signaling pathways-PKA and MAPK pathways. Previous study indicated that exogenous arginine could induce hyphal formation in several dimorphic fungi through hydrolysis by arginase, but inhibit the dimorphic transition of U. esculenta. We conducted this study to reveal the function of arginine on dimorphic transition of U. esculenta., Results: In this study, we found that arginine, but not its anabolites, could slow down the dimorphic transition of U. esculenta proportionally to the concentration of arginine. Besides, UeArginase, predicated coding arginase in U. esculenta was cloned and characterized. UeArginase mutants could actually increase the content of endogenous arginine, and slow down the dimorphic transition on either nutritious rich or poor medium. Either adding exogenous arginine or UeArginase deletion lead to down regulated expressions of UePkaC, UePrf1, mfa1.2, mfa2.1, pra1 and pra2, along with an increased content of arginine during mating process., Conclusion: Results of this study indicated a direct role of arginine itself on the inhibition of dimorphic transition of U. esculenta, independent of its hydrolysis by UeArginase.

Adaptation to the environment is a requirement for the survival of every organism. For pathogenic fungi this also implies coping with the different conditions that occur during the infection cycle. After detecting changes to external media, organisms must modify their gene expression patterns in order to accommodate the new circumstances. Control of gene expression is a complex process that involves the coordinated action of multiple regulatory elements. Chromatin modification is a well-known mechanism for controlling gene expression in response to environmental changes in all eukaryotes. In pathogenic fungi, chromatin modifications are known to play crucial roles in controlling host interactions and their virulence capacity, yet little is known about the specific genes they directly target and to which signals they respond. The smut fungus Ustilago maydis is an excellent model system in which multiple molecular and cellular approaches are available to study biotrophic interactions. Many target genes regulated during the infection process have been well studied, however, how they are controlled and specifically how chromatin modifications affect gene regulation in the context of infection is not well known in this organism. Here, we analyse the presence of chromatin modifying enzymes and complexes in U. maydis and discuss their putative roles in this plant pathogen in the context of findings from other organisms, including other plant pathogens such as Magnaporthe oryzae and Fusarium graminearum. We propose U. maydis as a remarkable organism with interesting chromatin features, which would allow finding new functions of chromatin modifications during plant pathogenesis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III 2 , and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O 2 and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III 2 and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rad51 serves to maintain and protect integrity of the genome through its actions in DNA repair and replication fork protection. The active form of Rad51 is a nucleoprotein filament consisting of chains of protomer units arranged linearly along single-stranded DNA. In a mutant screen using Ustilago maydis as an experimental system we identified a novel variant of Rad51, in which an amino acid change near the protomer-protomer interaction interface confers a strong trans dominant inhibitory effect on resistance to DNA damaging agents and proficiency in homologous recombination. Modeling studies of the mutated residue D161Y suggested that steric interference with surrounding residues was the likely cause of the inhibitory effect. Changes of two nearby residues, predicted from the modeling to minimize steric clashes, mitigated the inhibition of DNA repair. Direct testing of purified Rad51 D161Y protein in defined biochemical reactions revealed it to be devoid of DNA-binding activity itself, but capable of interfering with Rad51 WT in formation and maintenance of nucleoprotein filaments on single-stranded DNA and in DNA strand exchange. Rad51 D161Y protein appears to be unable to self-associate in solution and defective in forming complexes with the U. maydis BRCA2 ortholog., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.)

Ustilago maydis is a biotrophic fungus causing corn smut disease in maize. The secreted effector protein Pit2 is an inhibitor of papain-like cysteine proteases (PLCPs) essential for virulence. Pit2 inhibitory function relies on a conserved 14 amino acids motif (PID14). Here we show that synthetic PID14 peptides act more efficiently as PLCP inhibitors than the full-length Pit2 effector. Mass spectrometry shows processing of Pit2 by maize PLCPs, which releases an inhibitory core motif from the PID14 sequence. Mutational analysis demonstrates that two conserved residues are essential for Pit2 function. We propose that the Pit2 effector functions as a substrate mimicking molecule: Pit2 is a suitable substrate for apoplastic PLCPs and its processing releases the embedded inhibitor peptide, which in turn blocks PLCPs to modulate host immunity. Remarkably, the PID14 core motif is present in several plant associated fungi and bacteria, indicating the existence of a conserved microbial inhibitor of proteases (cMIP).

Objectives: Ustilago maydis lipase A (UMLA) expressed in Pichia pastoris was compared with Candida antarctica lipase A (CALA) to study its biochemical properties such as thermostability and selectivity., Results: UMLA had similar behavior to its homologue CALA regarding the effect of pH and temperature on enzymatic activity, substrate preference and selectivity. Both lipases were active on insoluble triglycerides as well as natural oils and hydrolyzed preferably esters with short and medium acyl and alkyl chains. Both enzymes were slightly selective for the (S)-glycidyl butyrate enantiomer and had a remarkable preference for the sn-2 position of triglycerides. The optimal activity was 40 and 50 °C for UMLA and CALA, respectively. However, temperature had a greater effect on the stability of UMLA compared to CALA, observing a half-life at 50 °C of 2.07 h and 12.83 h, respectively., Conclusions: UMLA shares some biochemical properties with CALA such as the sn-2 preference on triglyceride hydrolysis and transesterification. However, the high thermostability attributed to CALA was not observed in UMLA; this can be due to the lack of stabilization via AXXXA motifs in helices and fewer proline residues at the surface.

Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses 1,2 . The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway 3 . Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.

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.)

Fungalysins from several phytopathogenic fungi have been shown to be involved in cleavage of plant chitinases. While fungal chitinases are responsible for cell wall remodeling during growth and morphogenesis, plant chitinases are important components of immunity. This study describes a dual function of the Ustilago maydis fungalysin UmFly1 in modulation of both plant and fungal chitinases. Genetic, biochemical and microscopic experiments were performed to elucidate the in vitro and in planta functions of U. maydis UmFly1. U. maydis ∆umfly1 mutants show significantly reduced virulence, which coincides with reduced cleavage of the maize chitinase ZmChiA within its chitin-binding domain. Moreover, deletion of umfly1 affected the cell separation of haploid U. maydis sporidia. This phenotype is associated with posttranslational activation of the endogenous chitinase UmCts1. Genetic complementation of the ∆umfly1 mutant with a homologous gene from closely related, but nonpathogenic, yeast fully rescued the cell separation defect in vitro, but it could not recover the ∆umfly1 defect in virulence and cleavage of the maize chitinase. We report on the dual function of the secreted fungalysin UmFly1. We hypothesize that co-evolution of U. maydis with its host plant extended the endogenous function of UmFly1 towards the modulation of plant chitinase activity to promote infection., (© 2018 The Authors New Phytologist © 2018 New Phytologist Trust.)

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.

In most organisms, galactose is metabolized via the Leloir pathway, which is conserved from bacteria to mammals. Utilization of galactose requires a close interplay of the metabolic enzymes, as misregulation or malfunction of individual components can lead to the accumulation of toxic intermediate compounds. For the phytopathogenic basidiomycete Ustilago maydis, galactose is toxic for wildtype strains, i.e. leads to growth repression despite the presence of favorable carbon sources as sucrose. The galactose sensitivity can be relieved by two independent modifications: (1) by disruption of Hxt1, which we identify as the major transporter for galactose, and (2) by a point mutation in the gene encoding the galactokinase Gal1, the first enzyme of the Leloir pathway. The mutation in gal1(Y67F) leads to reduced enzymatic activity of Gal1 and thus may limit the formation of putatively toxic galactose-1-phosphate. However, systematic deletions and double deletions of different genes involved in galactose metabolism point to a minor role of galactose-1-phosphate in galactose toxicity. Our results show that molecular triggers for galactose toxicity in U. maydis differ from yeast and mammals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.)

Ustilago maydis is an aerobic basidiomycete that fully depends on oxidative phosphorylation for its supply of ATP, pointing to mitochondria as a key player in the energy metabolism of this organism. Mitochondrial F 1 F 0 -ATP synthase occurs in supramolecular structures. In this work, we isolated the monomer (640kDa) and the dimer (1280kDa) and characterized their subunit composition and kinetics of ATP hydrolysis. Mass spectrometry revealed that dimerizing subunits e and g were present in the dimer but not in the monomer. Analysis of the ATPase activity showed that both oligomers had Michaelis-Menten kinetics, but the dimer was 7 times more active than the monomer, while affinities were similar. The dimer was more sensitive to oligomycin inhibition, with a K i of 24nM, while the monomer had a K i of 169nM. The results suggest that the interphase between the monomers in the dimer state affects the catalytic efficiency of the enzyme and its sensitivity to inhibitors., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, mediates delivery of Rad51 to DNA during the course of homology-directed DNA repair. Rad51 interacts with Brh2 through the highly conserved BRC element and through a second region termed CRE located at the extreme carboxy terminus. Dss1, a small intrinsically unstructured protein that interacts with Brh2, is crucial for its activity in DNA repair, but the mechanism of this regulation is uncertain. In previous studies, we found that interaction of Brh2 with DNA was strongly modulated by association with Dss1. Here we report that CRE influences interaction of Dss1 with Brh2 and that Dss1 status markedly alters interaction of Brh2 with Rad51. While it appears that a single Rad51 protomer associates with Brh2 in complex with Dss1, loss of Dss1 is accompanied by a large increase in the number of Rad51 protomers that can associate with Brh2. Concomitant with this buildup of Rad51, Brh2 loses its ability to bind DNA. These observations suggest a feedback circuit in which release of Dss1 from Brh2 as it binds DNA triggers nucleation of a short Rad51 oligomer on Brh2, which in turn promotes dissociation of Brh2 from the DNA.

Ustilago maydis is an obligate biotrophic fungal pathogen which causes common smut disease of corn. To proliferate in host tissue, U. maydis must gain access to nutrients and overcome plant defence responses, such as the production of reactive oxygen species. The elucidation of the mechanisms by which U. maydis meets these challenges is critical for the development of strategies to combat smut disease. In this study, we focused on the contributions of phospholipases (PLs) to the pathogenesis of corn smut disease. We identified 11 genes encoding putative PLs and characterized the transcript levels for these genes in the fungus grown in culture and during infection of corn tissue. To assess the contributions of specific PLs, we focused on two genes, lip1 and lip2, which encode putative phospholipase A 2 (PLA 2 ) enzymes with similarity to platelet-activating factor acetylhydrolases. PLA 2 enzymes are known to counteract oxidative damage to lipids in other organisms. Consistent with a role in the mitigation of oxidative damage, lip2 mutants were sensitive to oxidative stress provoked by hydrogen peroxide and by increased production of reactive oxygen species caused by inhibitors of mitochondrial functions. Importantly, mutants defective in lip2, but not lip1, were attenuated for virulence in corn seedlings. Finally, a comparative analysis of fatty acid and cardiolipin profiles in the wild-type strain and a lip2 mutant revealed differences consistent with a protective role for Lip2 in maintaining lipid homeostasis and mitochondrial health during proliferation in the hostile host environment., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

Ustilago maydis, an edible mushroom growing on maize (Zea mays), is consumed as the food delicacy huitlacoche in Mexico. A chlorogenic acid esterase from this basidiomycete was expressed in good yields cultivating the heterologous host Pichia pastoris on the 5L bioreactor scale (reUmChlE; 45.9UL(-1)). In contrast to previously described chlorogenic acid esterases, the reUmChlE was also active towards feruloylated saccharides. The enzyme preferred substrates with the ferulic acid esterified to the O-5 position of arabinose residues, typical of graminaceous monocots, over the O-2 position of arabinose or the O-6 position of galactose residues. Determination of kcat/Km showed that the reUmChlE hydrolyzed chlorogenic acid 18-fold more efficiently than methyl ferulate, p-coumarate or caffeate. Phenolic acids were released by reUmChlE from natural substrates, such as destarched wheat bran, sugar beet pectin and coffee pulp. Treatment of wheat dough using reUmChlE resulted in a noticeable softening indicating a potential application of the enzyme in bakery and confectionery., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation.

Unlabelled: The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and β-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process., Importance: This study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

In the past decades our knowledge about fungal cell wall architecture increased tremendously and led to the identification of many enzymes involved in polysaccharide synthesis and remodeling, which are also of biotechnological interest. Fungal cell walls play an important role in conferring mechanic stability during cell division and polar growth. Additionally, in phytopathogenic fungi the cell wall is the first structure that gets into intimate contact with the host plant. A major constituent of fungal cell walls is chitin, a homopolymer of N-acetylglucosamine units. To ensure plasticity, polymeric chitin needs continuous remodeling which is maintained by chitinolytic enzymes, including lytic polysaccharide monooxygenases N-acetylglucosaminidases, and chitinases. Depending on the species and lifestyle of fungi, there is great variation in the number of encoded chitinases and their function. Chitinases can have housekeeping function in plasticizing the cell wall or can act more specifically during cell separation, nutritional chitin acquisition, or competitive interaction with other fungi. Although chitinase research made huge progress in the last decades, our knowledge about their role in phytopathogenic fungi is still scarce. Recent findings in the dimorphic basidiomycete Ustilago maydis show that chitinases play different physiological functions throughout the life cycle and raise questions about their role during plant-fungus interactions. In this work we summarize these functions, mechanisms of chitinase regulation and their putative role during pathogen/host interactions.

Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

The discovery of novel fungal lignocellulolytic enzymes is essential to improve the breakdown of plant biomass for the production of second-generation biofuels or biobased materials in green biorefineries. We previously reported that Ustilago maydis grown on maize secreted a diverse set of lignocellulose-acting enzymes including hemicellulases and putative oxidoreductases. One of the most abundant proteins of the secretome was a putative glucose-methanol-choline (GMC) oxidoreductase. The phylogenetic prediction of its function was hampered by the few characterized members within its clade. Therefore, we cloned the gene and produced the recombinant protein to high yield in Pichia pastoris. Functional screening using a library of substrates revealed that this enzyme was able to oxidize several aromatic alcohols. Of the tested aryl-alcohols, the highest oxidation rate was obtained with 4-anisyl alcohol. Oxygen, 1,4-benzoquinone, and 2,6-dichloroindophenol can serve as electron acceptors. This GMC oxidoreductase displays the characteristics of an aryl-alcohol oxidase (E.C.1.1.3.7), which is suggested to act on the lignin fraction in biomass.

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.)

Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Morphological changes are critical for host colonisation in plant pathogenic fungi. These changes occur at specific stages of their pathogenic cycle in response to environmental signals and are mediated by transcription factors, which act as master regulators. Histone deacetylases (HDACs) play crucial roles in regulating gene expression, for example by locally modulating the accessibility of chromatin to transcriptional regulators. It has been reported that HDACs play important roles in the virulence of plant fungi. However, the specific environment-sensing pathways that control fungal virulence via HDACs remain poorly characterised. Here we address this question using the maize pathogen Ustilago maydis. We find that the HDAC Hos2 is required for the dimorphic switch and pathogenic development in U. maydis. The deletion of hos2 abolishes the cAMP-dependent expression of mating type genes. Moreover, ChIP experiments detect Hos2 binding to the gene bodies of mating-type genes, which increases in proportion to their expression level following cAMP addition. These observations suggest that Hos2 acts as a downstream component of the cAMP-PKA pathway to control the expression of mating-type genes. Interestingly, we found that Clr3, another HDAC present in U. maydis, also contributes to the cAMP-dependent regulation of mating-type gene expression, demonstrating that Hos2 is not the only HDAC involved in this control system. Overall, our results provide new insights into the role of HDACs in fungal phytopathogenesis.

Complete utilization of all available carbon sources in lignocellulosic biomass still remains a challenge in engineering Saccharomyces cerevisiae. Even with efficient heterologous xylose catabolic pathways, S. cerevisiae is unable to utilize xylose in lignocellulosic biomass unless xylan is depolymerized to xylose. Here we demonstrate that a blended bioprospecting approach along with pathway engineering and evolutionary engineering can be used to improve xylan catabolism in S. cerevisiae. Specifically, we perform whole genome sequencing-based bioprospecting of a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. The heterologous expression of xylan catabolic genes enabled S. cerevisiae to grow on xylan as a single carbon source in minimal medium. A combination of bioprospecting and metabolic pathway evolution demonstrated that the xylan catabolic pathway could be further improved. Ultimately, engineering efforts were able to achieve xylan conversion into ethanol of up to 0.22 g/L on minimal medium compositions with xylan. This pathway provides a novel starting point for improving lignocellulosic conversion by yeast., (Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A recombinant xylanase gene (rxynUMB) from Ustilago maydis 521 was expressed in Pichia pastoris, and the enzyme was purified and characterized. Phylogenetic analysis demonstrated that rxynUMB belongs to glycosyl hydrolase family 11. The Trp84, Trp95, Glu93, and Glu189 residues are proposed to be present at the active site. The apparent molecular mass of the recombinant xylananse was approximately 24 kDa, and the optimum pH and temperature were 4.3 and 50 °C, respectively. Xylanase activity was enhanced by 166 and 115% with Fe(2+) and Mn(2+), respectively. The biochemical properties of this recombinant xylanase suggest that it may be a useful candidate for a variety of commercial applications.

Ustilago maydis, causal agent of corn smut disease, is a dimorphic fungus alternating between a saprobic budding haploid and an obligate pathogenic filamentous dikaryon. Maize responds to U. maydis colonization by producing tumorous structures, and only within these does the fungus sporulate, producing melanized sexual teliospores. Previously we identified Ust1, an APSES (Asm1p, Phd1p, Sok2p, Efg1p, and StuAp) transcription factor, whose deletion led to filamentous haploid growth and the production of highly pigmented teliospore-like structures in culture. In this study, we analyzed the transcriptome of a ust1 deletion mutant and functionally characterized two highly upregulated genes with potential roles in melanin biosynthesis: um05361, encoding a putative laccase (lac1), and um06414, encoding a polyketide synthase (pks1). The Δlac1 mutant strains showed dramatically reduced virulence on maize seedlings and fewer, less-pigmented teliospores in adult plants. The Δpks1 mutant was unaffected in seedling virulence but adult plant tumors generated hyaline, nonmelanized teliospores. Thus, whereas pks1 appeared to be restricted to the synthesis of melanin, lac1 showed a broader role in virulence. In conclusion, the ust1 deletion mutant provided an in vitro model for sporulation in U. maydis, and functional analysis supports the efficacy of this in vitro mutant analysis for identification of genes involved in in planta teliosporogenesis.

In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.

Brh2, the BRCA2 ortholog in the fungus Ustilago maydis, harbors two different DNA-binding domains, one located in the N-terminal region and the other located in the C-terminal region. Here we were interested in comparing the biochemical properties of Brh2 fragments, Brh2(NT) and Brh2(CT), respectively, harboring the two different DNA-binding regions to understand the mechanistic purpose of dual DNA-interaction domains. With oligonucleotide substrates to model different DNA conformations, it was found that the substrate specificity of Brh2(NT) and Brh2(CT) was almost indistinguishable although avidity was different depending on salt concentration. DNA annealing activity inherent in Brh2 was found to be attributable to Brh2(NT). Likewise, activity responsible for a second-end capture reaction modeling a later step in repair of DNA double-strand breaks was found attributable to Brh2(NT). Efficient annealing of DNA strands coated with RPA required full length Brh2 rather than Brh2(NT) suggesting Brh2(CT) contributes to the activity when RPA is present. Brh2(NT) and Brh2(CT) were both found capable of physically interacting with RPA. The results suggest that while the two DNA-binding regions of Brh2 appear functionally redundant in certain aspects of DNA repair, they differ in fundamental properties, and likely contribute in different ways to repair processes involving or arising from stalled DNA replication forks., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Many microorganisms secrete surface-active glycolipids. The basidiomycetous fungus Ustilago maydis produces two different classes of glycolipids, mannosylerythritol lipids (MEL) and ustilagic acids (UAs). Here we report that biosynthesis of MELs is partially localized in peroxisomes and coupled to peroxisomal fatty acid degradation. The acyltransferases, Mac1 and Mac2, which acylate mannosylerythritol with fatty acids of different length, contain a type 1 peroxisomal targeting signal (PTS1). We demonstrate that Mac1 and Mac2 are targeted to peroxisomes, while other enzymes involved in MEL production reside in different compartments. Mis-targeting of Mac1 and Mac2 to the cytosol did not block MEL synthesis but promoted production of MEL species with altered acylation pattern. This is in contrast to peroxisome deficient mutants that produced MELs similar to the wild type. We could show that cytosolic targeting of Mac1 and Mac2 reduces the amount of UA presumably due to competition for overlapping substrates. Interestingly, hydroxylated fatty acids characteristic for UAs appear in MELs corroborating cross-talk between both biosynthesis pathways. Therefore, peroxisomal localization of MEL biosynthesis is not only prerequisite for generation of the natural spectrum of MELs, but also facilitates simultaneous assembly of different glycolipids in a single cell., (© 2014 John Wiley & Sons Ltd.)

CalB of Pseudozyma aphidis (formerly named Candida antarctica) is one of the most widely applied enzymes in industrial biocatalysis. Here, we describe a protein with 66 % sequence identity to CalB, designated Ustilago maydis lipase 2 (Uml2), which was identified as the product of gene um01422 of the corn smut fungus U. maydis. Sequence analysis of Uml2 revealed the presence of a typical lipase catalytic triad, Ser-His-Asp with Ser125 located in a Thr-Xaa-Ser-Xaa-Gly pentapeptide. Deletion of the uml2 gene in U. maydis diminished the ability of cells to hydrolyse fatty acids from tributyrin or Tween 20/80 substrates, thus demonstrating that Uml2 functions as a lipase that may contribute to nutrition of this fungal pathogen. Uml2 was heterologously produced in Pichia pastoris and recombinant N-glycosylated Uml2 protein was purified from the culture medium. Purified Uml2 released short- and long-chain fatty acids from p-nitrophenyl esters and Tween 20/80 substrates. Furthermore, phosphatidylcholine substrates containing long-chain saturated or unsaturated fatty acids were effectively hydrolysed. Both esterase and phospholipase A activity of Uml2 depended on the Ser125 catalytic residue. These results indicate that Uml2, in contrast to CalB, exhibits not only esterase and lipase activity but also phospholipase A activity. Thus, by genome mining, we identified a novel CalB-like lipase with different substrate specificities.

Ustilago maydis is a pathogenic fungus that produces the corn smut. It is a biotrophic parasite that depends on living plant tissues for its proliferation and development. Polygalacturonases are secreted by pathogens to solubilize the plant cell-wall and are required for pathogen virulence. In this paper, we report the isolation of a U. maydis polygalacturonase gene (Pgu1) and the functional and structural characterization of the encoded enzyme. The U. maydis Pgu1 gene is expressed when the fungus is grown in liquid culture media containing different carbon sources. In plant tissue, the expression increased as a function of incubation time. Pgu1 gene expression was detected during plant infection around 10 days post-infection with U. maydis FB-D12 strain in combination with teliospore formation. Synthesis and secretion of active recombinant PGU1 were achieved using Pichia pastoris, the purified enzyme had a optimum temperature of 34 °C, optimum pH of 4.5, a Km of 57.84 g/L for polygalacturonic acid, and a Vmax of 28.9 µg/min mg. Structural models of PGU1 based on homologous enzymes yielded a typical right-handed β-helix fold of pectinolytic enzymes classified in the glycosyl hydrolases family 28, and the U. maydis PGU1 is related with endo rather than exo polygalacturonases., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Dipeptidyl aminopeptidases are enzymes involved in the posttranslational control of bioactive peptides. Here we identified the gene dapUm in Ustilago maydis by homology with other fungal dipeptidyl aminopeptidases. Analysis of the dapUm-deduced amino acid sequence indicated that it encodes for membrane-type serine protease with a characteristic prolyl oligopeptidase catalytic motif triad: Ser, Asp, His. In order to overexpress the DapUm, the gene encoding for it was cloned and transformed into Pichia. Using this system, we observed a ∼ 125-kDa recombinant protein with an optimal enzymatic activity at pH 6.0 and at 40 °C for the Ala-Pro-p-nitroanilide substrate and an experimental pH of 6.9. U. maydis DapUm was specifically inhibited by phenylmethylsulfonyl fluoride and Pefabloc, confirming the presence of a serine residue in the active site. To our knowledge, this study is the first report on the cloning and expression of a DPP IV dipeptidyl aminopeptidase from a basidiomycete organism. Moreover, the use of recombinant DapUm will allow us to further study and characterize this enzyme, in addition to testing chemical compounds for pharmaceutical purposes.

α-L-arabinofuranosidases are glycoside hydrolases that specifically hydrolyze non-reducing residues from arabinose-containing polysaccharides. In the case of arabinoxylans, which are the main components of hemicellulose, they are part of microbial xylanolytic systems and are necessary for complete breakdown of arabinoxylans. Glycoside hydrolase family 62 (GH62) is currently a small family of α-L-arabinofuranosidases that contains only bacterial and fungal members. Little is known about the GH62 mechanism of action, because only a few members have been biochemically characterized and no three-dimensional structure is available. Here, we present the first crystal structures of two fungal GH62 α-L-arabinofuranosidases from the basidiomycete Ustilago maydis (UmAbf62A) and ascomycete Podospora anserina (PaAbf62A). Both enzymes are able to efficiently remove the α-L-arabinosyl substituents from arabinoxylan. The overall three-dimensional structure of UmAbf62A and PaAbf62A reveals a five-bladed β-propeller fold that confirms their predicted classification into clan GH-F together with GH43 α-L-arabinofuranosidases. Crystallographic structures of the complexes with arabinose and cellotriose reveal the important role of subsites +1 and +2 for sugar binding. Intriguingly, we observed that PaAbf62A was inhibited by cello-oligosaccharides and displayed binding affinity to cellulose although no activity was observed on a range of cellulosic substrates. Bioinformatic analyses showed that UmAbf62A and PaAbf62A belong to two distinct subfamilies within the GH62 family. The results presented here provide a framework to better investigate the structure-function relationships within the GH62 family.

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)., (Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.)

Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; aminotransferase encoded by aspC, indole-3-pyruvic acid decarboxylase encoded by ipdC, and indole-3-acetic acid dehydrogenase encoded by iad1. The ipdC from Enterobacter cloacae ATCC 13047, aspC from Escherichia coli, and iad1 from Ustilago maydis were cloned and expressed under the control of the tac and sod promoters in E. coli. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expression under the control of P(tac), whereas AspC was efficiently expressed by P(sod) originating from Corynebacterium glutamicum. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli DH5α expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 h of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6- fold increase compared with wild-type DH5α harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 h cultivation in LB medium supplemented with 4 g/l of tryptophan.

Secreted fungal effectors mediate plant-fungus pathogenic interactions. These proteins are typically N-glycosylated, a common posttranslational modification affecting their location and function. N-glycosylation consists of the addition, and subsequent maturation, of an oligosaccharide core in the endoplasmic reticulum (ER) and Golgi apparatus. In this article, we show that two enzymes catalyzing specific stages of this pathway in maize smut (Ustilago maydis), glucosidase I (Gls1) and glucosidase II β-subunit (Gas2), are essential for its pathogenic interaction with maize (Zea mays). Gls1 is required for the initial stages of infection following appressorium penetration, and Gas2 is required for efficient fungal spreading inside infected tissues. While U. maydis Δgls1 cells induce strong plant defense responses, Δgas2 hyphae are able to repress them, showing that slight differences in the N-glycoprotein processing can determine the extent of plant-fungus interactions. Interestingly, the calnexin protein, a central element of the ER quality control system for N-glycoproteins in eukaryotic cells, is essential for avoiding plant defense responses in cells with defective N-glycoproteins processing. Thus, N-glycoprotein maturation and this conserved checkpoint appear to play an important role in the establishment of an initial biotrophic state with the plant, which allows subsequent colonization.

The fungal and plant plasma membrane H⁺-ATPases play critical roles in the physiology of yeast, plant and protozoa cells. We identified two genes encoding two plasma membrane H⁺-ATPases in the basidiomycete Ustilago maydis, one protein with higher identity to fungal (um02581) and the other to plant (um01205) H⁺-ATPases. Proton pumping activity was 5-fold higher when cells were grown in minimal medium with ethanol compared to cells cultured in rich YPD medium, but total vanadate-sensitive ATPase activity was the same in both conditions. In contrast, the activity in cells cultured in minimal medium with glucose was 2-fold higher than in YPD or ethanol, implicating mechanisms for the regulation of the plasma membrane ATPase activity in U. maydis. Analysis of gene expression of the H⁺-ATPases from cells grown under different conditions, showed that the transcript expression of um01205 (plant-type) was higher than that of um02581 (fungal-type). The translation of the two proteins was confirmed by mass spectrometry analysis. Unlike baker's yeast and plant H⁺-ATPases, where the activity is increased by a short incubation with glucose or sucrose, respectively, U. maydis H⁺-ATPase activity did not change in response to these sugars. Sequence analysis of the two U. maydis H⁺-ATPases revealed the lack of canonical threonine and serine residues which are targets of protein kinases in Saccharomyces cerevisiae and Arabidopsis thaliana plasma membrane H⁺-ATPases, suggesting that phosphorylation of the U. maydis enzymes occurs at different amino acid residues.

The sustainable control of basidiomycete biotrophic plant pathogenesis requires an understanding of host responses to infection, as well as the identification and functional analysis of fungal genes involved in disease development. The creation and analysis of a suppressive subtractive hybridization (SSH) cDNA library from Ustilago maydis-infected Zea mays seedlings enabled the identification of fungal and plant genes expressed during disease development, and uncovered new insights into the interactions of this model system. Candidate U. maydis pathogenesis genes were identified by using the current SSH cDNA library analysis, and by knowledge generated from previous cDNA microarray and comparative genomic analyses. These identifications were supported by the independent determination of transcript level changes in different cell-types and during pathogenic development. The basidiomycete specific um01632, the highly in planta expressed um03046 (zig1), and the calcineurin regulatory B subunit (um10226, cnb1), were chosen for deletion experiments. um01632 and zig1 mutants showed no difference in morphology and did not have a statistically significant impact on pathogenesis. cnb1 mutants had a distinct cell division phenotype and reduced virulence in seedling assays. Infections with reciprocal wild-type×Δcnb1 haploid strain crosses revealed that the wild-type allele was unable to fully compensate for the lack of a second cnb1 allele. This haploinsufficiency was undetected in other fungal cnb1 mutational analyses. The reported data improves U. maydis genome annotation and expands on the current understanding of pathogenesis genes in this model basidiomycete., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The utilization of raw biomass components such as cellulose or hemicellulose for the production of valuable chemicals has attracted considerable research interest in recent years. One promising approach is the application of microorganisms that naturally convert biomass constituents into value added chemicals. One of these organisms--Ustilago maydis--can grow on xylan, the second most abundant polysaccharide in nature, while at the same time it produces chemicals of biotechnological interest., Results: In this study, we present the identification of an endo-1,4-beta xylanase responsible for xylan degradation. Xylanase activity of U. maydis cells was indirectly detected by the quantification of released reducing sugars and could be confirmed by visualizing oligosaccharides as degradation products of xylan by thin layer chromatography. A putative endo-1,4-beta-xylanase, encoded by um06350.1, was identified in the supernatant of xylan-grown cells. To confirm the activity, we displayed the putative xylanase on the surface of the xylanase negative Saccharomyces cerevisiae EBY100. The presented enzyme converted xylan to xylotriose, similar to the source organism U. maydis., Conclusions: The xylan degradation ability together with its unicellular and yeast-like growth makes U. maydis MB215 a promising candidate for the production of valuable chemicals such as itaconic acid or glycolipids from lignocellulosic biomass. Therefore, the characterization of the endo-1,4-beta-xylanase, encoded by um06350.1, is a further step towards the biotechnological application of U. maydis and its enzymes.

Salicylic acid (SA) is a key plant defence hormone which plays an important role in local and systemic defence responses against biotrophic pathogens like the smut fungus Ustilago maydis. Here we identified Shy1, a cytoplasmic U. maydis salicylate hydroxylase which has orthologues in the closely related smuts Ustilago hordei and Sporisorium reilianum. shy1 is transcriptionally induced during the biotrophic stages of development but not required for virulence during seedling infection. Shy1 activity is needed for growth on plates with SA as a sole carbon source. The trigger for shy1 transcriptional induction is SA, suggesting the possibility of a SA sensing mechanism in this fungus., (© 2013 John Wiley & Sons Ltd.)

Components of the cAMP (cyclic AMP) signalling cascades are conserved from fungi to humans, and are particularly important for fungal dimorphism and pathogenicity. Previous work has described two phosphodiesterases, UmPde1 and UmPde2, in Ustilago maydis which show strong phosphodiesterase activity. We further characterized the biological function(s) of these phosphodiesterases in U. maydis. Specifically, we examined their possible role(s) in regulation of the cAMP-dependent protein kinase A (PKA) pathway and their roles in filamentous growth and pathogenicity. We found that UmPde1, which shares 35 % similarity with Cryptococcus neoformans Pde1, also displays functional homology with this enzyme. UmPde1 complements the capsule-formation defect of C. neoformans strains deleted for Pde1. In U. maydis, the cell morphology of the umpde1 deletion mutant resembled the multiple budding phenotypes seen with the ubc1 mutant, which lacks the regulatory subunit of PKA. Interestingly, on low-ammonium medium, umpde2 deletion strains showed a reduction in filamentation that was comparable to that of ubc1 deletion strains; however, umpde1 deletion strains showed normal filamentation on low-ammonium medium. Furthermore, both the ubc1 deletion strain in which the PKA pathway was constitutively active and the umpde1 deletion strains were significantly reduced in pathogenicity, while the umpde2 deletion strains showed a trend for reduced pathogenicity compared with wild-type strains. These data support a role for the phosphodiesterases UmPde1 and UmPde2 in regulating the U. maydis cAMP-dependent PKA pathway through modulation of cAMP levels, thus affecting dimorphic growth and pathogenicity.

The morphogenesis-related NDR kinase (MOR) pathway regulates morphogenesis in fungi. In spite of the high conservation of its components, impairing their functions results in highly divergent cellular responses depending on the fungal species. The reasons for such differences are unclear. Here we propose that the species-specific connections between cell cycle regulation and the MOR pathway could be partly responsible for these divergences. We based our conclusion on the characterization of the MOR pathway in the fungus Ustilago maydis. Each gene that encodes proteins of this pathway in U. maydis was deleted. All mutants exhibited a constitutive hyperpolarized growth, contrasting with the loss of polarity observed in other fungi. Using a conditional allele of the central NDR kinase Ukc1, we found that impairing MOR function resulted in a prolonged G2 phase. This cell cycle delay appears to be the consequence of an increase in Cdk1 inhibitory phosphorylation. Strikingly, prevention of the inhibitory Cdk1 phosphorylation abolished the hyperpolarized growth associated with MOR pathway depletion. We found that the prolonged G2 phase resulted in higher levels of expression of crk1, a conserved kinase that promotes polar growth in U. maydis. Deletion of crk1 also abolished the dramatic activation of polar growth in cells lacking the MOR pathway. Taken together, our results suggest that Cdk1 inhibitory phosphorylation may act as an integrator of signaling cascades regulating fungal morphogenesis and that the distinct morphological response observed in U. maydis upon impairment of the MOR pathway could be due to a cell cycle deregulation.

A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzymatically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from U. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal glycosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catabolism of β-1,3-glucan after its release in the extracellular medium.