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Characterization of the Largest Effector Gene Cluster of Ustilago maydis  [PDF]
Thomas Brefort equal contributor,Shigeyuki Tanaka equal contributor,Nina Neidig equal contributor,Gunther Doehlemann equal contributor,Volker Vincon,Regine Kahmann
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1003866
Abstract: In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.
Identification of O-mannosylated Virulence Factors in Ustilago maydis  [PDF]
Alfonso Fernández-álvarez,Miriam Marín-Menguiano,Daniel Lanver,Alberto Jiménez-Martín,Alberto Elías-Villalobos,Antonio J. Pérez-Pulido,Regine Kahmann,José I. Ibeas
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002563
Abstract: The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.
Plant Surface Cues Prime Ustilago maydis for Biotrophic Development  [PDF]
Daniel Lanver equal contributor,Patrick Berndt equal contributor,Marie Tollot,Vikram Naik,Miroslav Vranes,Tobias Warmann,Karin Münch,Nicole R?ssel,Regine Kahmann
PLOS Pathogens , 2014, DOI: doi/10.1371/journal.ppat.1004272
Abstract: Infection-related development of phytopathogenic fungi is initiated by sensing and responding to plant surface cues. This response can result in the formation of specialized infection structures, so-called appressoria. To unravel the program inducing filaments and appressoria in the biotrophic smut fungus Ustilago maydis, we exposed cells to a hydrophobic surface and the cutin monomer 16-hydroxy hexadecanoic acid. Genome-wide transcriptional profiling at the pre-penetration stage documented dramatic transcriptional changes in almost 20% of the genes. Comparisons with the U. maydis sho1 msb2 double mutant, lacking two putative sensors for plant surface cues, revealed that these plasma membrane receptors regulate a small subset of the surface cue-induced genes comprising mainly secreted proteins including potential plant cell wall degrading enzymes. Targeted gene deletion analysis ascribed a role to up-regulated GH51 and GH62 arabinofuranosidases during plant penetration. Among the sho1/msb2-dependently expressed genes were several secreted effectors that are essential for virulence. Our data also demonstrate specific effects on two transcription factors that redirect the transcriptional regulatory network towards appressorium formation and plant penetration. This shows that plant surface cues prime U. maydis for biotrophic development.
Gene discovery and transcript analyses in the corn smut pathogen Ustilago maydis: expressed sequence tag and genome sequence comparison
Eric CH Ho, Matt J Cahill, Barry J Saville
BMC Genomics , 2007, DOI: 10.1186/1471-2164-8-334
Abstract: Four cDNA libraries were constructed using RNA isolated from U. maydis diploid teliospores (U. maydis strains 518 × 521) and haploid cells of strain 521 grown under nutrient rich, carbon starved, and nitrogen starved conditions. Using the genome sequence as a scaffold, the 15,901 ESTs were assembled into 6,101 contiguous expressed sequences (contigs); among these, 5,482 corresponded to predicted genes in the MUMDB (MIPS Ustilago maydis database), while 619 aligned to regions of the genome not yet designated as genes in MUMDB. A comparison of EST abundance identified numerous genes that may be regulated in a cell type or starvation-specific manner. The transcriptional response to nitrogen starvation was assessed using RT-qPCR. The results of this suggest that there may be cross-talk between the nitrogen and carbon signalling pathways in U. maydis. Bioinformatic analysis identified numerous examples of alternative splicing and anti-sense transcription. While intron retention was the predominant form of alternative splicing in U. maydis, other varieties were also evident (e.g. exon skipping). Selected instances of both alternative splicing and anti-sense transcription were independently confirmed using RT-PCR.Through this work: 1) substantial sequence information has been provided for U. maydis genome annotation; 2) new genes were identified through the discovery of 619 contigs that had previously escaped annotation; 3) evidence is provided that suggests the regulation of nitrogen metabolism in U. maydis differs from that of other model fungi, and 4) Alternative splicing and anti-sense transcription were identified in U. maydis and, amid similar observations in other basidiomycetes, this suggests these phenomena may be widespread in this group of fungi. These advances emphasize the importance of EST analysis in genome annotation.Ustilago maydis is a ubiquitous pathogen of Zea mays (corn) that can cause tremendous economic losses [1]. The most conspicuous symptom of the
Efecto de la concanavalina a en el cultivo del fitopatógeno ustilago maydis
Saavedra Hernández,Elsa; Pérez Santiago,Alma; Pérez Campos,Eduardo; Córdoba Alva,Félix;
Interciencia , 2003,
Abstract: the effect of concanavalin a on ustilago maydis cultures was tested in order to gain understanding about plant-fungi infection mechanisms. growth kinetics of u. maydis in culture indicates that con a promoted discrete culture growth after 9h of incubation, as compared with fungi cultures devoid of the lectin. binding of con a to basidial cells induced morphological changes such as agglutination, branching, ramification and increased budding. the use of a fluorescent conjugate of the lectin (alexa-fluor) revealed points of increased fluorescence on the basidial cell tips and budding zones, suggesting a heterogeneous distribution of receptors on the cell wall surface during growth. the addition of a-d-methyl mannopyranoside to these preparations occluded the specific fluorescence.
Biomass pretreatment affects Ustilago maydis in producing itaconic acid
Tobias Klement, Sofia Milker, Gernot J?ger, Philipp M Grande, Pablo Domínguez de María, Jochen Büchs
Microbial Cell Factories , 2012, DOI: 10.1186/1475-2859-11-43
Abstract: U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.Since fossil fuels are limited, many current research projects are investigating the utilization of renewable resources to ensure the sustainable production of biofuels and platform chemicals. Recently, most of these approaches have focused on producing alcohols from starch which competes with the food supply chain. Moreover, these approaches waste most of the plant biomass. Thus, new research is focusing on utilizing ligno-cellulose as the prime raw material for biofuel production [1] and constructing new biocatalysts for this purpose [2].Itaconic acid (C5H6O4, methylene su
Pep1, a Secreted Effector Protein of Ustilago maydis, Is Required for Successful Invasion of Plant Cells  [PDF]
Gunther Doehlemann,Karina van der Linde,Daniela A?mann,Daniela Schwammbach,Alexander Hof,Amitabh Mohanty,David Jackson,Regine Kahmann
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000290
Abstract: The basidiomycete Ustilago maydis causes smut disease in maize. Colonization of the host plant is initiated by direct penetration of cuticle and cell wall of maize epidermis cells. The invading hyphae are surrounded by the plant plasma membrane and proliferate within the plant tissue. We identified a novel secreted protein, termed Pep1, that is essential for penetration. Disruption mutants of pep1 are not affected in saprophytic growth and develop normal infection structures. However, Δpep1 mutants arrest during penetration of the epidermal cell and elicit a strong plant defense response. Using Affymetrix maize arrays, we identified 116 plant genes which are differentially regulated in Δpep1 compared to wild type infections. Most of these genes are related to plant defense. By in vivo immunolocalization, live-cell imaging and plasmolysis approaches, we detected Pep1 in the apoplastic space as well as its accumulation at sites of cell-to-cell passages. Site-directed mutagenesis identified two of the four cysteine residues in Pep1 as essential for function, suggesting that the formation of disulfide bridges is crucial for proper protein folding. The barley covered smut fungus Ustilago hordei contains an ortholog of pep1 which is needed for penetration of barley and which is able to complement the U. maydis Δpep1 mutant. Based on these results, we conclude that Pep1 has a conserved function essential for establishing compatibility that is not restricted to the U. maydis / maize interaction.
The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis  [PDF]
Kai Heimel equal contributor,Mario Scherer equal contributor,Miroslav Vranes,Ramon Wahl,Chetsada Pothiratana,David Schuler,Volker Vincon,Florian Finkernagel,Ignacio Flor-Parra,J?rg K?mper
PLOS Pathogens , 2010, DOI: 10.1371/journal.ppat.1001035
Abstract: In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development.
Activation of the Cell Wall Integrity Pathway Promotes Escape from G2 in the Fungus Ustilago maydis  [PDF]
Natalia Carbó,José Pérez-Martín
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1001009
Abstract: It is widely accepted that MAPK activation in budding and fission yeasts is often associated with negative effects on cell cycle progression, resulting in delay or arrest at a specific stage in the cell cycle, thereby enabling cells to adapt to changing environmental conditions. For instance, activation of the Cell Wall Integrity (CWI) pathway in the budding yeast Saccharomyces cerevisiae signals an increase in CDK inhibitory phosphorylation, which leads cells to remain in the G2 phase. Here we characterized the CWI pathway of Ustilago maydis, a fungus evolutionarily distant from budding and fission yeasts, and show that activation of the CWI pathway forces cells to escape from G2 phase. In spite of these disparate cell cycle responses in S. cerevisiae and U. maydis, the CWI pathway in both organisms appears to respond to the same class cell wall stressors. To understand the basis of such a difference, we studied the mechanism behind the U. maydis response. We found that activation of CWI pathway in U. maydis results in a decrease in CDK inhibitory phosphorylation, which depends on the mitotic phosphatase Cdc25. Moreover, in response to activation of the CWI pathway, Cdc25 accumulates in the nucleus, providing a likely explanation for the increase in the unphosphorylated form of CDK. We also found that the extended N-terminal domain of Cdc25, which is dispensable under normal growth conditions, is required for this G2 escape as well as for resistance to cell wall stressors. We propose that the process of cell cycle adaptation to cell stress evolved differently in these two divergent organisms so that each can move towards a cell cycle phase most appropriate for responding to the environmental signals encountered.
Compatibility in the Ustilago maydis–Maize Interaction Requires Inhibition of Host Cysteine Proteases by the Fungal Effector Pit2  [PDF]
André N. Mueller,Sebastian Ziemann,Steffi Treitschke ,Daniela A?mann,Gunther Doehlemann
PLOS Pathogens , 2013, DOI: 10.1371/journal.ppat.1003177
Abstract: The basidiomycete Ustilago maydis causes smut disease in maize, with large plant tumors being formed as the most prominent disease symptoms. During all steps of infection, U. maydis depends on a biotrophic interaction, which requires an efficient suppression of plant immunity. In a previous study, we identified the secreted effector protein Pit2, which is essential for maintenance of biotrophy and induction of tumors. Deletion mutants for pit2 successfully penetrate host cells but elicit various defense responses, which stops further fungal proliferation. We now show that Pit2 functions as an inhibitor of a set of apoplastic maize cysteine proteases, whose activity is directly linked with salicylic-acid-associated plant defenses. Consequently, protease inhibition by Pit2 is required for U. maydis virulence. Sequence comparisons with Pit2 orthologs from related smut fungi identified a conserved sequence motif. Mutation of this sequence caused loss of Pit2 function. Consequently, expression of the mutated protein in U. maydis could not restore virulence of the pit2 deletion mutant, indicating that the protease inhibition by Pit2 is essential for fungal virulence. Moreover, synthetic peptides of the conserved sequence motif showed full activity as protease inhibitor, which identifies this domain as a new, minimal protease inhibitor domain in plant-pathogenic fungi.
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