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From the evening of March 12, till dinner on March 13, 2017, the 1st International Ustilago/Smut Convergence took place as a workshop prior to the start of the 29th Fungal Genetics Conference, in Asilomar, California. The overall goals of the meeting were to expand the smut model systems being used and to expand participation by the next generations of scientists with these fungi. These goals were implemented through a combination of emphasis on student and post-doc presentations, mentoring of such individuals, and active recruitment of participation by groups under-represented at such meetings in recent years in the US, especially those from Latin America and other Spanish-speaking countries. Work was presented at the first workshop on U. maydis, Sporosorium reilianum, Microbotryum violaceum, U. esculenta, and Thecaphora thlaspeos. Students and post-doctoral researchers were encouraged to present their "just-in-time," as-yet-unpublished data, in a safe environment, with the understanding of those attending the meeting that this early access was a privilege not to be taken advantage of. The result was lively and constructive discussion, including a variety of presentations by these young scientists on putative and characterized smut effector proteins, clearly at the forefront of such research, even considering the advances presented later that week at the Fungal Genetics Conference. This review also briefly compares the first meeting with the events of the recent 2nd International Ustilago/Smut Convergence (March 11-12, 2019), which ended with a tribute to Prof. Dr. Regine Kahmann, in honor of her career, and especially for her contributions to the field of smut genetics., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Biotrophic fungal plant pathogens cause billions of dollars in losses to North American crops annually. The model for functional investigation of these fungi is Ustilago maydis. Its 20.5 Mb annotated genome sequence has been an excellent resource for investigating biotrophic plant pathogenesis. Expressed-sequence tag libraries and microarray hybridizations have provided insight regarding the type of transcripts produced by U. maydis but these analyses were not comprehensive and there were insufficient data for transcriptome comparison to other smut fungi. To improve transcriptome annotation and enable comparative analyses, comprehensive strand-specific RNA-seq was performed on cell-types of three related smut species: U. maydis (common smut of corn), Ustilago hordei (covered smut of barley), and Sporisorium reilianum (head smut of corn)., Results: In total, >1 billion paired-end sequence reads were obtained from haploid cell, dikaryon and teliospore RNA of U. maydis, haploid cell RNA of U. hordei, and haploid and dikaryon cell RNA of S. reilianum. The sequences were assembled into transfrags using Trinity, and updated gene models were created using PASA and categorized with Cufflinks Cuffcompare. Representative genes that were predicted for the first time with these RNA-seq analyses and genes with novel annotation features were independently assessed by reverse transcriptase PCR. The analyses indicate hundreds more predicted proteins, relative to the previous genome annotation, could be produced by U. maydis from altered transcript forms, and that the number of non-coding RNAs produced, including transcribed intergenic sequences and natural antisense transcripts, approximately equals the number of mRNAs. This high representation of non-coding RNAs appears to be a conserved feature of the smut fungi regardless of whether they have RNA interference machinery. Approximately 50% of the identified NATs were conserved among the smut fungi., Conclusions: Overall, these analyses revealed: 1) smut genomes encode a number of transcriptional units that is twice the number of annotated protein-coding genes, 2) a small number of intergenic transcripts may encode proteins with characteristics of fungal effectors, 3) the vast majority of intergenic and antisense transcripts do not contain ORFs, 4) a large proportion of the identified antisense transcripts were detected at orthologous loci among the smut fungi, and 5) there is an enrichment of functional categories among orthologous loci that suggests antisense RNAs could have a genome-wide, non-RNAi-mediated, influence on gene expression in smut fungi.

The basidiomycete smut fungus Ustilago maydis causes common smut of corn. This disease is spread through the production of teliospores, which are thick-walled dormant structures characterized by low rates of respiration and metabolism. Teliospores are formed when the fungus grows within the plant, and the morphological steps involved in their formation have been described, but the molecular events leading to dormancy are not known. In U. maydis, natural antisense transcripts (NATs) can function to alter gene expression and many NATs have increased levels in the teliospore. One such NAT is as-ssm1 which is complementary to the gene for the mitochondrial seryl-tRNA synthetase (ssm1), an enzyme important to mitochondrial function. The disruption of ssm1 leads to cell lysis, indicating it is also essential for cellular viability. To assess the function of as-ssm1, it was ectopically expressed in haploid cells, where it is not normally present. This expression led to reductions in growth rate, virulence, mitochondrial membrane potential and oxygen consumption. It also resulted in the formation of as-ssm1/ssm1 double-stranded RNA and increased ssm1 transcript levels, but no change in Ssm1 protein levels was detected. Together, these findings suggest a role for as-ssm1 in facilitating teliospore dormancy through dsRNA formation and reduction of mitochondrial function., (© 2016 John Wiley & Sons Ltd.)

In this study, Ustilago maydis Ndt80 homolog one, unh1, of the obligate sexual pathogen U. maydis,is described. Unh1 is the sole Ndt80-like DNA-binding protein inU. maydis. In this model basidiomycete, Unh1 plays a role in sexual development, influencing tumor maturation, teliospore development and subsequent meiotic completion. Teliospore formation was reduced in deletion mutants, and those that did form had unpigmented, hyaline cell walls, and germinated without completing meiosis. Constitutively expressing unh1 in haploid cells resulted in abnormal pigmentation, when grown in both potato dextrose broth and minimal medium, suggesting that pigmentation may be triggered by unh1 in U. maydis. The function of Unh1 in sexual development and pigment production depends on the presence of the Ndt80-like DNA-binding domain, identified within Unh1. In the absence of this domain, or when the binding domain was altered with targeted amino acid changes, ectopic expression of Unh1 failed to complement the unh1 deletion with regards to pigment production and sexual development. An investigation of U. maydis genes with upstream motifs similar to Ndt80 recognition sequences revealed that some have altered transcript levels in Δunh1 strains. We propose that the first characterized Ndt80-like DNA-binding protein in a basidiomycete, Unh1, acts as a transcription factor that is required for teliospore maturation and the completion of meiosis in U. maydis., (Copyright © 2016. Published by Elsevier Inc.)

Ustilago maydis is the causative agent of common smut of corn. Early studies noted its ability to synthesize phytohormones and, more recently these growth promoting substances were confirmed as cytokinins (CKs). Cytokinins comprise a group of phytohormones commonly associated with actively dividing tissues. Lab analyses identified variation in virulence between U. maydis dikaryon and solopathogen infections of corn cob tissue. Samples from infected cob tissue were taken at sequential time points post infection and biochemical profiling was performed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS). This hormone profiling revealed that there were altered levels of ABA and major CKs, with a marked reduction in CK glucosides, increases in methylthiol CKs and a particularly dramatic increase in cisZ CK forms, in U. maydis infected tissue. These changes were more pronounced in the more virulent dikaryon relative to the solopathogenic strain suggesting a role for cytokinins in moderating virulence during biotrophic infection. These findings highlight the fact that U. maydis does not simply mimic a fertilized seed but instead reprograms the host tissue. Results underscore the suitability of the Ustilago maydis- Zea mays model as a basis for investigating the control of phytohormone dynamics during biotrophic infection of plants.

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.)

Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis., (© 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Ustilago maydis is the model for investigating basidiomycete biotrophic plant pathogens. To further the annotation of its genome, 12,943 full-length cDNA sequences were used to construct databases for the promoter and untranslated regions of U. maydis genes. A subset of clones was sequenced to determine full cDNA sequences. These and the original ESTs were assembled into contigs representing 3,058, or 45%, of the predicted U. maydis genes. The new sequencing allowed the confirmation of 2,842 gene models, 690 of which contain an intron. The use of full-length cDNA clone sequences ensured that untranslated regions were physically linked to the open reading frames (ORFs), not merely aligned upstream of the start of transcription. Identified sequence features include: (1) over 500 potential short upstream ORFs, (2) 95 gene models that require further annotation, (3) one new potential ORF, (4) varying GC content in different gene regions, (5) a WebLogo motif for the start of translation, (6) the correlation of UTR length with transcript representation in cDNA libraries and with gene function categories, (7) a relationship between natural antisense transcripts and UTR length that differs from that of Saccharomyces cerevisiae, (8) a potential relationship between DNA replication and the control of transcription, and (9) new insights regarding mechanisms for the control of transcription and mRNA maturation in U. maydis.

In the corn smut pathogen, Ustilago maydis, meiosis and teliospore germination are temporally linked. We review teliospore dormancy and germination in U. maydis and present an overview of meiosis in basidiomycetes. The relevant available expressed sequence tag data is discussed, the databases used in reciprocal best hit blastp analysis are presented and potential U. maydis meiosis genes are identified. The implications of identifying these genes are discussed and hypotheses are presented regarding the control of meiosis in U. maydis.

Background: Ustilago maydis is the basidiomycete fungus responsible for common smut of corn and is a model organism for the study of fungal phytopathogenesis. To aid in the annotation of the genome sequence of this organism, several expressed sequence tag (EST) libraries were generated from a variety of U. maydis cell types. In addition to utility in the context of gene identification and structure annotation, the ESTs were analyzed to identify differentially abundant transcripts and to detect evidence of alternative splicing and anti-sense transcription., Results: Four cDNA libraries were constructed using RNA isolated from U. maydis diploid teliospores (U. maydis strains 518 x 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., Conclusion: 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 maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Ustilago maydis is a model fungal pathogen that induces the formation of tumors in maize. The tumor provides an environment for hyphal differentiation, leading to the formation of thick-walled, diploid teliospores. Such spores serve as a dispersal agent for smut and rust fungi, and their germination leads to new rounds of infection. The morphological changes that occur during teliospore germination in U. maydis have been described in detail. However, the specific molecular events that facilitate this process have not been identified. Through the construction and hybridization of microarrays containing a set of 3918 non-redundant cDNAs, we have identified genes that are differentially regulated during teliospore germination. Teliospores induced to germinate for 4 and 11 h were selected for comparison with dormant teliospores. Genes identified as differentially expressed included many that are presumably involved in as yet undescribed molecular events during teliospore germination, as well as characterized genes previously shown to be required for the process. This study represents the first large-scale investigation of changes in gene expression during teliospore germination.

When fungi interact with plants as pathogens or as symbionts, there are often changes in fungal cell morphology and nuclear state. This study establishes the use of cDNA microarrays to detect gene expression changes in Ustilago maydis cells that differ in structure and nuclear content. Categorizing differentially expressed genes on the basis of function indicated that U. maydis cell types vary most in the expression of genes related to metabolism. We also observed that more genes are up-regulated in the filamentous dikaryon than in the filamentous diploid, relative to non-pathogenic budding cells. Our comparison of pathogenic development indicated that the dikaryon is more virulent than the diploid. Other identified expression patterns suggest a cell-specific difference in nutrient acquisition, cell metabolism and signal transduction. The relevance of gene expression change to cell type biology is discussed.

A large-scale comparative genomic analysis of unisequence sets obtained from an Ustilago maydis EST collection was performed against publicly available EST and genomic sequence datasets from 21 species. We annotated 70% of the collection based on similarity to known sequences and recognized protein signatures. Distinct grouping of the ESTs, defined by the presence or absence of similar sequences in the species examined, allowed the identification of U. maydis sequences present only (1) in fungal species, (2) in plants but not animals, (3) in animals but not plants, or (4) in all three eukaryotic lineages assessed. We also identified 215 U. maydis genes that are found in the ascomycete but not in the basidiomycete genome sequences searched. Candidate genes were identified for further functional characterization. These include 167 basidiomycete-specific sequences, 58 fungal pathogen-specific sequences (including 37 basidiomycete pathogen-specific sequences), and 18 plant pathogen-specific sequences, as well as two sequences present only in other plant pathogen and plant species.

Ustilago maydis is an important model system for the plant pathogenic smut and rust fungi. Critical to the continued development of this model is establishing genomic resources. We have constructed a cDNA library from a forced diploid culture of U. maydis growing as filaments and have generated 7455 ESTs that are assembled into 3074 contiguous sequences. This represents as much as 46% of the coding capacity predicted for U. maydis. BLAST searches with a similarity cutoff of E

Ustilago maydis grows in its host Zea mays eliciting the formation of obvious tumors that are full of black teliospores. Teliospores are thick-walled, dormant, diploid cells that have evolved for dispersal and survival of the pathogen. Their germination leads to new rounds of infection and is temporally linked to meiosis. We are investigating gene expression during teliospore germination to gain insight into the control of this process. Here we identify genes expressed through creation of an expressed sequence tag (EST) library. We generated 2871 ESTs that are assembled into 1293 contiguous sequences. Based upon a blast search similarity cutoff of E < or =10(-5) 38% of all contigs were orphans while 62% showed similarity to sequences in the protein database. Analyses of blast searches were used to functionally classify genes. Northern hybridizations using specific cDNA clones reveal a relative level of expression consistent with the number of sequences per contig. Identified genes and expression information provide a base for genome annotation of U. maydis and further investigation of teliospore germination and pathogenesis.