Novel Secreted Effectors Conserved Among Smut Fungi Contribute to the Virulence of Ustilago maydis.

Fungal pathogens deploy a set of molecules (proteins, specialized metabolites, and sRNAs), so-called effectors, to aid the infection process. In comparison to other plant pathogens, smut fungi have small genomes and secretomes of 20 Mb and around 500 proteins, respectively. Previous comparative genomic studies have shown that many secreted effector proteins without known domains, i.e., novel, are conserved only in the Ustilaginaceae family. By analyzing the secretomes of 11 species within Ustilaginaceae, we identified 53 core homologous groups commonly present in this lineage. By collecting existing mutants and generating additional ones, we gathered 44 Ustilago maydis strains lacking single core effectors as well as 9 strains containing multiple deletions of core effector gene families. Pathogenicity assays revealed that 20 of these 53 mutant strains were affected in virulence. Among the 33 mutants that had no obvious phenotypic changes, 13 carried additional, sequence-divergent, structurally similar paralogs. We report a virulence contribution of seven previously uncharacterized single core effectors and of one effector family. Our results help to prioritize effectors for understanding U. maydis virulence and provide genetic resources for further characterization. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Positively Selected Effector Genes and Their Contribution to Virulence in the Smut Fungus Sporisorium reilianum.

Plants and fungi display a broad range of interactions in natural and agricultural ecosystems ranging from symbiosis to parasitism. These ecological interactions result in coevolution between genes belonging to different partners. A well-understood example is secreted fungal effector proteins and their host targets, which play an important role in pathogenic interactions. Biotrophic smut fungi (Basidiomycota) are well-suited to investigate the evolution of plant pathogens, because several reference genomes and genetic tools are available for these species. Here, we used the genomes of Sporisorium reilianum f. sp. zeae and S. reilianum f. sp. reilianum, two closely related formae speciales infecting maize and sorghum, respectively, together with the genomes of Ustilago hordei, Ustilago maydis, and Sporisorium scitamineum to identify and characterize genes displaying signatures of positive selection. We identified 154 gene families having undergone positive selection during species divergence in at least one lineage, among which 77% were identified in the two investigated formae speciales of S. reilianum. Remarkably, only 29% of positively selected genes encode predicted secreted proteins. We assessed the contribution to virulence of nine of these candidate effector genes in S. reilianum f. sp. zeae by deleting individual genes, including a homologue of the effector gene pit2 previously characterized in U. maydis. Only the pit2 deletion mutant was found to be strongly reduced in virulence. Additional experiments are required to understand the molecular mechanisms underlying the selection forces acting on the other candidate effector genes, as well as the large fraction of positively selected genes encoding predicted cytoplasmic proteins.

A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem.

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover, our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver.

A Tale of Genome Compartmentalization: The Evolution of Virulence Clusters in Smut Fungi.

Smut fungi are plant pathogens mostly parasitizing wild species of grasses as well as domesticated cereal crops. Genome analysis of several smut fungi including Ustilago maydis revealed a singular clustered organization of genes encoding secreted effectors. In U. maydis, many of these clusters have a role in virulence. Reconstructing the evolutionary history of clusters of effector genes is difficult because of their intrinsically fast evolution, which erodes the phylogenetic signal and homology relationships. Here, we describe the use of comparative evolutionary analyses of quality draft assemblies of genomes to study the mechanisms of this evolution. We report the genome sequence of a South African isolate of Sporisorium scitamineum, a smut fungus parasitizing sugar cane with a phylogenetic position intermediate to the two previously sequenced species U. maydis and Sporisorium reilianum. We show that the genome of S. scitamineum contains more and larger gene clusters encoding secreted effectors than any previously described species in this group. We trace back the origin of the clusters and find that their evolution is mainly driven by tandem gene duplication. In addition, transposable elements play a major role in the evolution of the clustered genes. Transposable elements are significantly associated with clusters of genes encoding fast evolving secreted effectors. This suggests that such clusters represent a case of genome compartmentalization that restrains the activity of transposable elements on genes under diversifying selection for which this activity is potentially beneficial, while protecting the rest of the genome from its deleterious effect.

Genome comparison of barley and maize smut fungi reveals targeted loss of RNA silencing components and species-specific presence of transposable elements.

Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maize-infecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts.

FGDB: revisiting the genome annotation of the plant pathogen Fusarium graminearum.

The MIPS Fusarium graminearum Genome Database (FGDB) was established as a comprehensive genome database on one of the most devastating fungal plant pathogens of wheat, barley and maize. The current version of FGDB v3.1 provides information on the full manually revised gene set based on the Broad Institute assembly FG3 genome sequence. The results of gene prediction tools were integrated with the help of comparative data on related species to result in a set of 13.718 annotated protein coding genes. This rigorous approach involved adding or modifying gene models and represents a coding sequence gold standard for the genus Fusarium. The gene loci improvements results in 2461 genes which either are new or have different structures compared to the Broad Institute assembly 3 gene set. Moreover the database serves as a convenient entry point to explore expression data results and to obtain information on the Affymetrix GeneChip probe sets. The resource is accessible on http://mips.gsf.de/genre/proj/FGDB/.

Pathogenicity determinants in smut fungi revealed by genome comparison.

Biotrophic pathogens, such as the related maize pathogenic fungi Ustilago maydis and Sporisorium reilianum, establish an intimate relationship with their hosts by secreting protein effectors. Because secreted effectors interacting with plant proteins should rapidly evolve, we identified variable genomic regions by sequencing the genome of S. reilianum and comparing it with the U. maydis genome. We detected 43 regions of low sequence conservation in otherwise well-conserved syntenic genomes. These regions primarily encode secreted effectors and include previously identified virulence clusters. By deletion analysis in U. maydis, we demonstrate a role in virulence for four previously unknown diversity regions. This highlights the power of comparative genomics of closely related species for identification of virulence determinants.

Tandem KH domains of Khd4 recognize AUACCC and are essential for regulation of morphology as well as pathogenicity in Ustilago maydis.

RNA-binding proteins constitute key factors of the post-transcriptional machinery. These regulatory proteins recognize specific elements within target transcripts to promote, for example, maturation, translation, or stability of mRNAs. In Ustilago maydis, evidence is accumulating that post-transcriptional processes are important to determine pathogenicity. Deletion of khd4, encoding a predicted RNA-binding protein with five K homology (KH) domains, causes aberrant cell morphology and reduced virulence. Here, we demonstrate that Khd4 recognizes the sequence AUACCC in vivo via its tandem KH domains 3 and 4. This sequence most likely functions as a regulatory RNA element in U. maydis, since it accumulates in 3' untranslated regions. Consistently, an independent mRNA expression profiling approach revealed that the binding motif is significantly enriched in transcripts showing altered expression levels in khd4Delta strains. Since the vast majority of potential Khd4 target mRNAs exhibit increased amounts in deletion mutants, Khd4 might promote mRNA instability. Mutants that fail to bind AUACCC resemble deletion mutants, which exhibit altered cell morphology, disturbed filamentous growth, and severely reduced virulence. Hence, RNA binding is essential for function of Khd4, stressing the importance of post-transcriptional control in regulating morphology and pathogenicity.

Relationship between phylogenetic distribution and genomic features in Neurospora crassa.

In the post-genome era, insufficient functional annotation of predicted genes greatly restricts the potential of mining genome data. We demonstrate that an evolutionary approach, which is independent of functional annotation, has great potential as a tool for genome analysis. We chose the genome of a model filamentous fungus Neurospora crassa as an example. Phylogenetic distribution of each predicted protein coding gene (PCG) in the N. crassa genome was used to classify genes into six mutually exclusive lineage specificity (LS) groups, i.e. Eukaryote/Prokaryote-core, Dikarya-core, Ascomycota-core, Pezizomycotina-specific, N. crassa-orphans and Others. Functional category analysis revealed that only approximately 23% of PCGs in the two most highly lineage-specific grouping, Pezizomycotina-specific and N. crassa-orphans, have functional annotation. In contrast, approximately 76% of PCGs in the remaining four LS groups have functional annotation. Analysis of chromosomal localization of N. crassa-orphan PCGs and genes encoding for secreted proteins showed enrichment in subtelomeric regions. The origin of N. crassa-orphans is not known. We found that 11% of N. crassa-orphans have paralogous N. crassa-orphan genes. Of the paralogous N. crassa-orphan gene pairs, 33% were tandemly located in the genome, implying a duplication origin of N. crassa-orphan PCGs in the past. LS grouping is thus a useful tool to explore and understand genome organization, evolution and gene function in fungi.

The posttranscriptional machinery of Ustilago maydis.

Eukaryotic gene expression begins with transcription and maturation of mRNAs in the nucleus and ends with their translation and degradation in the cytoplasm. Here, we present an inventory of the posttranscriptional machinery of Ustilago maydis that is based on the recently sequenced genome and its comprehensive manual annotation. We used the detailed knowledge available for Saccharomyces cerevisiae and higher eukaryotes to predict posttranscriptional components in this plant pathogen. The comparison to S. cerevisiae revealed that most core components are shared. Both fungi belong to the small group of organisms lacking components of the RNAi machinery. However, a striking difference exists at the level of splicing. U. maydis harbors substantially more intron-containing genes and this correlates with the presence of numerous splice components with human orthologues that are absent or less conserved in S. cerevisiae. In particular, U. maydis contains three out of four core proteins of the exon junction complex, which marks spliced exons and is involved in cytoplasmic mRNA transport. In this context, it is also remarkable that the U. maydis genome displays components involved in microtubule- rather than actin-dependent mRNA transport. Thus, U. maydis might serve as an attractive model system to gain novel insights into posttranscriptional processes.

Genomic evolution of the proteasome system among hemiascomycetous yeasts.

Components of the proteasome-ubiquitin pathway are highly conserved throughout eukaryotic organisms. In S. cerevisiae, the expression of proteasomal genes is subject to concerted control by a transcriptional regulator, Rpn4p, interacting with a highly conserved cis-regulatory element, PACE, located in the upstream regions of these genes. Taking advantage of sequence data accumulated from 15 Hemiascomycetes, we performed an in silico study to address the problem of how this system might have evolved among these species. We found that in all these species the Rpn4p homologues are well conserved in terms of sequence and characteristic domain features. The "PACE patterns" turned out to be nearly identical among the Saccharomyces "sensu stricto" species, whereas in the evolutionary more distant species the putatively functional cis-regulatory motifs revealed deviations from the "canonical" PACE nonamere sequence in one or two nucleotides. Our findings suggest that during evolution of the Hemiascomycetes such slightly divergent ancestral motifs have converged into a unique PACE element for the majority of the proteasomal genes within the most recent species of this class. Likewise, the Rpn4 factors within the most recent species of this class show a higher degree of similarity in sequence than their ancestral counterparts. By contrast, we did not detect PACE-like motifs among the proteasomal genes in other eukaryotes, such as S. pombe, several filamentous fungi, A. thaliana, or humans, leaving the interesting question which type of concerted regulation of the proteasome system has developed in species other than the Hemiascomycetes.

The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization.

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher rates of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.

FGDB: a comprehensive fungal genome resource on the plant pathogen Fusarium graminearum.

The MIPS Fusarium graminearum Genome Database (FGDB) is a comprehensive genome database on one of the most devastating fungal plant pathogens of wheat and barley. FGDB provides information on two gene sets independently derived by automated annotation of the F.graminearum genome sequence. A complete manually revised gene set will be completed within the near future. The initial results of systematic manual correction of gene calls are already part of the current gene set. The database can be accessed to retrieve information from bioinformatics analyses and functional classifications of the proteins. The data are also organized in the well established MIPS catalogs and novel query techniques are available to search the data. The comprehensive set of gene calls was also used for the design of an Affymetrix GeneChip. The resource is accessible on http://mips.gsf.de/genre/proj/fusarium/.

Long-oligomer microarray profiling in Neurospora crassa reveals the transcriptional program underlying biochemical and physiological events of conidial germination.

To test the inferences of spotted microarray technology against a biochemically well-studied process, we performed transcriptional profiling of conidial germination in the filamentous fungus, Neurospora crassa. We first constructed a 70 base oligomer microarray that assays 3366 predicted genes. To estimate the relative gene expression levels and changes in gene expression during conidial germination, we analyzed a circuit design of competitive hybridizations throughout a time course using a Bayesian analysis of gene expression level. Remarkable consistency of mRNA profiles with previously published northern data was observed. Genes were hierarchically clustered into groups with respect to their expression profiles over the time course of conidial germination. A functional classification database was employed to characterize the global picture of gene expression. Consensus motif searches identified a putative regulatory component associated with genes involved in ribosomal biogenesis. Our transcriptional profiling data correlate well with biochemical and physiological processes associated with conidial germination and will facilitate functional predictions of novel genes in N.crassa and other filamentous ascomycete species. Furthermore, our dataset on conidial germination allowed comparisons to transcriptional mechanisms associated with germination processes of diverse propagules, such as teliospores of the phytopathogenic fungus Ustilago maydis and spores of the social amoeba Dictyostelium discoideum.

The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes.

In this paper, we present the Functional Catalogue (FunCat), a hierarchically structured, organism-independent, flexible and scalable controlled classification system enabling the functional description of proteins from any organism. FunCat has been applied for the manual annotation of prokaryotes, fungi, plants and animals. We describe how FunCat is implemented as a highly efficient and robust tool for the manual and automatic annotation of genomic sequences. Owing to its hierarchical architecture, FunCat has also proved to be useful for many subsequent downstream bioinformatic applications. This is illustrated by the analysis of large-scale experiments from various investigations in transcriptomics and proteomics, where FunCat was used to project experimental data into functional units, as 'gold standard' for functional classification methods, and also served to compare the significance of different experimental methods. Over the last decade, the FunCat has been established as a robust and stable annotation scheme that offers both, meaningful and manageable functional classification as well as ease of perception.

Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism.

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence.

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.

Large scale analysis of sequences from Neurospora crassa.

After 50 years of analysing Neurospora crassa genes one by one large scale sequence analysis has increased the number of accessible genes tremendously in the last few years. Being the only filamentous fungus for which a comprehensive genomic sequence database is publicly accessible N. crassa serves as the model for this important group of microorganisms. The MIPS N. crassa database currently holds more than 16 Mb of non-redundant data of the chromosomes II and V analysed by the German Neurospora Genome Project. This represents more than one-third of the genome. Open reading frames (ORFs) have been extracted from the sequence and the deduced proteins have been annotated extensively. They are classified according to matches in sequence databases and attributed to functional categories according to their relatives. While 41% of analysed proteins are related to known proteins, 30% are hypothetical proteins with no match to a database entry. The entire genome is expected to comprise some 13000 protein coding genes, more than twice as many as found in yeasts, and reflects the high potential of filamentous fungi to cope with various environmental conditions.