The monokaryotic filamentous fungus Ustilago sp. HFJ311 promotes plant growth and reduces Cd accumulation by enhancing Fe transportation and auxin biosynthesis.

Infection with smut fungus like Ustilago maydis decreases crop yield via inducing gall formation. However, the in vitro impact of Ustilago spp. on plant growth and stress tolerance remains elusive. This study investigated the plant growth promotion and cadmium stress mitigation mechanisms of a filamentous fungus discovered on a cultural medium containing 25 µM CdCl2. ITS sequence alignment revealed 98.7 % similarity with Ustilago bromivora, naming the strain Ustilago sp. HFJ311 (HFJ311). Co-cultivation with HFJ311 significantly enhanced the growth of various plants, including Arabidopsis, tobacco, cabbage, carrot, rice, and maize, and improved Arabidopsis tolerance to abiotic stresses like salt and metal ions. HFJ311 increased chlorophyll and Fe contents in Arabidopsis shoots and enhanced root-to-shoot Fe translocation while decreasing root Fe concentration by approximately 70 %. Concurrently, HFJ311 reduced Cd accumulation in Arabidopsis by about 60 %, indicating its potential for bioremediation in Cd-contaminated soils. Additionally, HFJ311 stimulated IAA concentration by upregulating auxin biosynthesis genes. Overexpression of the Fe transporter IRT1 negated HFJ311's growth-promotion effects under Cd stress. These results suggest that HFJ311 stimulates plant growth and inhibits Cd uptake by enhancing Fe translocation and auxin biosynthesis while disrupting Fe absorption. Our findings offer a promising bioremediation strategy for sustainable agriculture and food security.

Establishing an itaconic acid production process with Ustilago species on the low-cost substrate starch.

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.

Comparative transcriptomics reveal different mechanisms for hyphal growth across four plant-associated dimorphic fungi.

Fungal dimorphism is a phenomenon by which a fungus can grow both as a yeast form and a hyphal form. It is frequently related to pathogenicity as different growth forms are more suitable for different functions during a life cycle. Among dimorphic plant pathogens, the corn smut fungus Ustilago maydis serves as a model organism to understand fungal dimorphism and its effect on pathogenicity. However, there is a lack of data on whether mechanisms elucidated from model species are broadly applicable to other fungi. In this study, two non-model plant-associated species in the smut fungus subphylum (Ustilaginomycotina), Tilletiopsis washingtonensis and Meira miltonrushii, were selected to compare dimorphic mechanisms in these to those in U. maydis. We sequenced transcriptomic profiles during both yeast and hyphal growth in these two species using Tween40, a lipid mimic, as a trigger for hyphal growth. We then compared our data with previously published data from U. maydis and a fourth but unrelated dimorphic phytopathogen, Ophiostoma novo-ulmi. Comparative transcriptomics was performed to identify common genes upregulated during hyphal growth in all four dimorphic species. Intriguingly, T. washingtonensis shares the least similarities of transcriptomic alteration (hyphal growth versus yeast growth) with the others, although it is closely related to M. miltonrushii and U. maydis. This suggests that phylogenetic relatedness is not correlated with transcriptomic similarity under the same biological phenomenon. Among commonly expressed genes in the four species, genes in cell energy production and conversion, amino acid transport and metabolism and cytoskeleton are significantly enriched. Considering dimorphism genes characterized in U. maydis, as well as hyphal tip-associated genes from the literature, we found only genes encoding the cell end marker Tea4/TeaC and the kinesin motor protein Kin3 concordantly expressed in all four species. This suggests a divergence in species-specific mechanisms for dimorphic transition and hyphal growth.

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

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

Cloning and disruption of the UeArginase in Ustilago esculenta: evidence for a role of arginine in its dimorphic transition.

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.

A potent chitin-hydrolyzing enzyme from Myrothecium verrucaria affects growth and development of Helicoverpa armigera and plant fungal pathogens.

Chitin, a crucial structural and functional component of insects and fungi, serves as a target for pest management by utilizing novel chitinases. Here, we report the biocontrol potential of recombinant Myrothecium verrucaria endochitinase (rMvEChi) against insect pest and fungal pathogens. A complete ORF of MvEChi (1185 bp) was cloned and heterologously expressed in Escherichia coli. Structure based sequence alignment of MvEChi revealed the presence of conserved domains SXGG and DXXDXDXE specific for GH-18 family, involved in substrate binding and catalysis, respectively. rMvEChi (46.6 kDa) showed optimum pH and temperature as 7.0 and 30 °C, respectively. Furthermore, rMvEChi remained stable within the pH range of 6.0 to 8.0 and up to 40 °C. rMvEChi exhibited kcat/Km values of 129.83 × 103 [(g/L)-1 s-1] towards 4MU chitotrioside. Hydrolysis of chitooligosaccharides with various degrees of polymerization (DP) using rMvEChi indicated the release of DP2 as main end product with order of reaction as DP6 > DP5 > DP4 > DP3. Bioassay of rMvEChi against Helicoverpa armigera displayed potent anti-feedant activity and induced mortality. In vitro antifungal activity against plant pathogenic fungi (Ustilago maydis and Bipolaris sorokiniana) exhibited significant inhibition of mycelium growth. These results suggest that MvEChi has significant potential in enzyme-based pest and pathogen management.

Mating-type loci of Ustilago esculenta are essential for mating and development.

Ustilago esculenta is closely related to the smut fungus Ustilago maydis and, in an endophytic-like life in the plant Zizania latifolia, only infects host stems and causes swollen stems to form edible galls called Jiaobai in China. In order to study its different modes of invasion and sites of symptom development from other smut fungi at the molecular level, we first characterized the a and b mating-type loci of U. esculenta. The a loci contained three a mating-type alleles, encoding two pheromones and one pheromone receptor per allele. The pheromone/receptor system controlled the conjugation formation, the initial step of mating, in which each pheromone was specific for recognition by only one mating partner. In addition, there are at least three b alleles identified in U. esculenta, encoding two subunits of heterodimeric homeodomain transcription factors bE and bW, responsible for hyphal growth and invasiveness. Hyphal formation, elongation and invasion after mating of two compatible partners occurred, only when a heterodimer complex was formed by the bE and bW proteins derived from different alleles. We also demonstrated that even with only one paired pheromone-pheromone receptor, the active b locus heterodimer triggered hyphal growth and infection.

Physicochemical, thermal, and rheological properties of nixtamalized blue-corn flours and masas added with huitlacoche (Ustilago maydis) paste.

The aim of this work was to evaluate the effect of the addition of huitlacoche paste to nixtamalized blue-corn flours (NBCF) on the physicochemical, thermal, and rheological properties of masas. Raw blue maize was nixtamalized (hydrothermal alkalinized process), then was wet-milled in a stone mill, masa was dehydrated, pulverized and sieved to obtain NBCF; commercial nixtamalized blue-corn flour (CNBCF) was used as a control. Huitlacoche paste in concentrations of 3, 6, 9, 12, 15, and 18% was added to nixtamalized flours. Characteristics of the blue grain showed its great effects on water absorption, viscosity, and masa cohesiveness; the addition of huitlacoche significantly influenced adhesiveness, water-absorption, color, and the rheological properties (p < 0.05). Values between 0.03 and 0.083 kg-force resulted in masas with optimal adhesiveness. The inclusion of huitlacoche paste can be achieved with a maximal addition of 9% in NBCF for an industrial process and could comprise a new industrialization alternative.

Identification of a novel member of the pH responsive pathway Pal/Rim in Ustilago maydis.

The most important signal transduction mechanism related to environmental pH responses in fungi is the Pal/Rim pathway. Our knowledge of this pathway came initially from studies on Ascomycota species where it is made by seven members divided into two complexes, one located at the plasma membrane, and other at the endosomal membrane. In Basidiomycota sepecies only the homologs of the endosomal membrane complex (genes PalA/Rim20, PalB/ Rim13, and PalC/ Rim23), plus the transcription factor PacC/Rim101 have been identified. In this study, we describe the identification in Ustilago maydis of a gene encoding a Rho-like protein (tentatively named RHO4) as a novel member of this pathway. The RHO4 gene possibly plays, among other functions, a role in the second proteolytic cleavage that leads to the activation of the transcription factor PacC/Rim101. Mutants in this gene showed a pleiotropic phenotype, displaying similar characteristics to the Pal/Rim mutants, such as a lower growth rate at alkaline pH, high sensitivity to ionic and osmotic stresses, and impairment in protease secretion, but no alteration of the yeast-to-mycelium dimorphic transition induced by acid pH whereas it has a function in the dimorphic transition induced by fatty acids.

UmTea1, a Kelch and BAR domain-containing protein, acts at the cell cortex to regulate cell morphogenesis in the dimorphic fungus Ustilago maydis.

The spatial organization of a cell is crucial for distribution of cell components and for cell morphogenesis in all organisms. Ustilago maydis, a basidiomycete fungus, has a yeast-like and a filamentous form. The former buds once per cell cycle at one of the cell poles, and can use the same site repeatedly or choose a new site at the same pole or opposite pole. The filamentous form consists of a long apical cell with short septate basal compartments lacking cytoplasm. It grows at the apex and can reverse growth forming a new growth zone at the basal end. We are interested in understanding how these different morphologies are generated. Here we present identification and characterization of U. maydis Tea1, a homologue of the fission yeast cell end marker Tea1. We demonstrate that UmTea1, a Kelch domain protein, interacts with itself and is an important determinant of the site of polarized growth: tea1 mutants bud simultaneously from both cell poles and form bifurcate buds. UmTea1 also regulates septum positioning, cell wall deposition, cell and neck width, coordination of nuclear division and cell separation, and localization of sterol-rich membrane domains. Some of these functions are shared with UmTea4, another cell end marker. We show that Tea1::GFP localizes to sites of polarized or potential polarized growth and to the septation site in the yeast-like form. Additionally, localization of Tea1::GFP as rings along the filament suggests that the filament undergoes septation. We hypothesize that Tea1 may act as a scaffold for the assembly of proteins that determine the site of polarized growth.

Virulence function of the Ustilago maydis sterol carrier protein 2.

The peroxisomal sterol carrier protein 2 (Scp2) of the biotrophic maize pathogen Ustilago maydis was detected in apoplastic fluid, suggesting that it might function as a secreted effector protein. Here we analyze the role of the scp2 gene during plant colonization. We used reverse genetics approaches to delete the scp2 gene, determined stress sensitivity and fatty acid utilization of mutants, demonstrated secretion of Scp2, used quantitative reverse transcription polymerase chain reaction for expression analysis and expressed GFP-Scp2 fusion proteins for protein localization. scp2 mutants were strongly attenuated in virulence and this defect manifested itself during penetration. Scp2 localized to peroxisomes and peroxisomal targeting was necessary for its virulence function. Deletion of scp2 in U. maydis interfered neither with growth nor with peroxisomal ß-oxidation. Conventionally secreted Scp2 protein could not rescue the virulence defect. scp2 mutants displayed an altered localization of peroxisomes. Our results show a virulence function for Scp2 during penetration that is probably carried out by Scp2 in peroxisomes. We speculate that Scp2 affects the lipid composition of membranes and in this way ensures the even cellular distribution of peroxisomes.

Coordinate regulation of Ustilago maydis ammonium transporters and genes involved in mating and pathogenicity.

The dimorphic switch from budding to filamentous growth is an essential morphogenetic transition many fungi utilize to cause disease in the host. Although different environmental signals can induce filamentous growth, the developmental programs associated with transmitting these different signals may differ. Here, we explore the relationship between filamentation and expression levels of ammonium transporters (AMTs) that also sense low ammonium for Ustilago maydis, the pathogen of maize. Overexpression of the high affinity ammonium transporter, Ump2, under normally non-inducing conditions, results in filamentous growth. Furthermore, ump2 expression levels are correlated with expression of genes involved in the mating response pathway and in pathogenicity. Ump1 and Ump2 transcription levels also tracked expression of genes normally up-regulated during either filamentous growth or during growth of the fungus inside the host. Interestingly, haploid strains deleted for the b mating-type locus, like those deleted for ump2, failed to filament on low ammonium; they also shared some alterations in gene expression patterns with cells deleted for ump2 or over-expressing this gene. Deletion of ump2 either in both mating partners or in a solopathogenic haploid strain resulted in a dramatic reduction in disease severity for infected plants, suggesting some importance of this transceptor in the pathogenesis program.

Cloning and characterization of the UePrf1 gene in Ustilago esculenta.

Ustilago esculenta, an obligate parasite of Zizania latifolia, is a typical dimorphic fungus which induces host stem swelling and inhibits host inflorescence development, but is not found in host leaves. Previous studies have shown that dimorphic switching is essential for fungal pathogenicity and is regulated by protein kinase A and mitogen-activated protein kinase (MAPK) signaling pathways that are integrated by Prf1 in Ustilago maydis. In this study we identified a Prf1 homolog in U. esculenta, designated UePrf1, encoding 830 amino acids with a conserved high mobility group domain located between amino acids 124 and 195. UePrf1 was upregulated during the mating process, which induces dimorphism in U. esculenta. In vitro, UePrf1 mutants showed defects in the mating process, including cell fusion and hyphal growth. UePrf1 mutants also show reduced expression of a genes, even during the cell fusion process. Additionally, the defect in hyphal growth of the UeKpp2 and UeKpp6 mutants (MAPK signaling pathway mutants) was partially counteracted by UePrf1 overexpression, along with induced b gene expression. These results provide evidence that UePrf1 is a key factor coordinating dimorphism in U. esculenta and suggest a conserved role for UePrf1 in the regulation of the a and b genes.

The Biotrophic Development of Ustilago maydis Studied by RNA-Seq Analysis.

The maize smut fungus Ustilago maydis is a model organism for elucidating host colonization strategies of biotrophic fungi. Here, we performed an in depth transcriptional profiling of the entire plant-associated development of U. maydis wild-type strains. In our analysis, we focused on fungal metabolism, nutritional strategies, secreted effectors, and regulatory networks. Secreted proteins were enriched in three distinct expression modules corresponding to stages on the plant surface, establishment of biotrophy, and induction of tumors. These modules are likely the key determinants for U. maydis virulence. With respect to nutrient utilization, we observed that expression of several nutrient transporters was tied to these virulence modules rather than being controlled by nutrient availability. We show that oligopeptide transporters likely involved in nitrogen assimilation are important virulence factors. By measuring the intramodular connectivity of transcription factors, we identified the potential drivers for the virulence modules. While known components of the b-mating type cascade emerged as inducers for the plant surface and biotrophy module, we identified a set of yet uncharacterized transcription factors as likely responsible for expression of the tumor module. We demonstrate a crucial role for leaf tumor formation and effector gene expression for one of these transcription factors.

Transcriptional analysis of the adaptation of Ustilago maydis during growth under nitrogen fixation conditions.

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 N2 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 N2 fixing bacterial endosymbiont is a flexible process that may be active during the adaptation of the fungus to the different nitrogen sources.

The ESCRT regulator Did2 maintains the balance between long-distance endosomal transport and endocytic trafficking.

In highly polarised cells, like fungal hyphae, early endosomes function in both endocytosis as well as long-distance transport of various cargo including mRNA and protein complexes. However, knowledge on the crosstalk between these seemingly different trafficking processes is scarce. Here, we demonstrate that the ESCRT regulator Did2 coordinates endosomal transport in fungal hyphae of Ustilago maydis. Loss of Did2 results in defective vacuolar targeting, less processive long-distance transport and abnormal shuttling of early endosomes. Importantly, the late endosomal protein Rab7 and vacuolar protease Prc1 exhibit increased shuttling on these aberrant endosomes suggesting defects in endosomal maturation and identity. Consistently, molecular motors fail to attach efficiently explaining the disturbed processive movement. Furthermore, the endosomal mRNP linker protein Upa1 is hardly present on endosomes resulting in defects in long-distance mRNA transport. In conclusion, the ESCRT regulator Did2 coordinates precise maturation of endosomes and thus provides the correct membrane identity for efficient endosomal long-distance transport.

Ustilago maydis, the corn smut fungus, has an unusual diploid mitotic stage.

Ustilago maydis causes common smut disease in maize. Although pathogenic diploid strains of the fungus have been known for many years, the normal life cycle was thought to involve an extended dikaryotic stage, with nuclear fusion occurring in immature teliospores. However, microscopic examination of both living and fixed tumor material showed that nuclei fuse long before sporulation begins and that tumors are filled with uninucleate cells undergoing mitosis. Quantification of DNA in the nuclei confirmed these observations. Additionally, fungal cells from tumor material placed on nutrient agar produced colonies of diploid budding cells. Time-lapse observations showed that at least some of these colonies arose from thin-walled fungal cells rather than from immature spores. Ultrastructural examination of developing teliospores from tumors confirmed that they were uninucleate. Condensed chromatin and other structures characteristic of nuclei in prophase I of meiosis were observed. These observations support revising the U. maydis life cycle to include a diploid mitotic stage that corresponds with rapid tumor enlargement and conversion of plant to fungal biomass. Because mitotic division of diploid nuclei is so unusual as a life cycle feature in the fungi, it will be interesting to explore the consequences of its presence in U. maydis.

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

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

Spatial organization of organelles in fungi: Insights from mathematical modelling.

Mathematical modelling in cellular systems aims to describe biological processes in a quantitative manner. Most accurate modelling is based on robust experimental data. Here we review recent progress in the theoretical description of motor behaviour, early endosome motility, ribosome distribution and peroxisome transport in the fungal model system Ustilago maydis and illustrate the power of modelling in our quest to understand molecular details and cellular roles of membrane trafficking in filamentous fungi.

UmTco1, a Hybrid Histidine Kinase Gene, Is Essential for the Sexual Development and Virulence of Ustilago maydis.

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.