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Plant pathogenic fungi colonizing living plant tissue secrete a cocktail of effector proteins to suppress plant immunity and reprogramme host cells. Although many of these effectors function inside host cells, delivery systems used by pathogenic bacteria to translocate effectors into host cells have not been detected in fungi. Here, we show that five unrelated effectors and two membrane proteins from Ustilago maydis, a biotrophic fungus causing smut disease in corn, form a stable protein complex. All seven genes appear co-regulated and are only expressed during colonization. Single mutants arrest in the epidermal layer, fail to suppress host defence responses and fail to induce non-host resistance, two reactions that likely depend on translocated effectors. The complex is anchored in the fungal membrane, protrudes into host cells and likely contacts channel-forming plant plasma membrane proteins. Constitutive expression of all seven complex members resulted in a surface-exposed form in cultured U. maydis cells. As orthologues of the complex-forming proteins are conserved in smut fungi, the complex may become an interesting fungicide target., This study reports that five effectors and two transmembrane proteins from the plant pathogenic fungus Ustilago maydis form a stable cell surface-exposed protein complex required for virulence.

The biotrophic fungus Ustilago maydis causes the smut disease of maize. The interaction with its host and induction of characteristic tumors are governed largely by secreted effectors whose function is mostly unknown. To identify effectors with a prominent role in virulence, we used RNA sequencing and found that the gene sta1 is upregulated during early stages of infection. We characterized Sta1 by comparative genomics, reverse genetics, protein localization, stress assays, and microscopy. sta1 mutants show a dramatic reduction of virulence and show altered colonization of tissue neighboring the vascular bundles. Functional orthologues of Sta1 are found in related smut pathogens infecting monocot and dicot plants. Sta1 is secreted by budding cells but is attached to the cell wall of filamentous hyphae. Upon constitutive expression of Sta1, fungal filaments become susceptible to Congo red, β-glucanase, and chitinase, suggesting that Sta1 alters the structure of the fungal cell wall. Constitutive or delayed expression of sta1 during plant colonization negatively impacts on virulence. Our results suggest that Sta1 is a novel kind of effector, which needs to modify the hyphal cell wall to allow hyphae to be accommodated in tissue next to the vascular bundles.

Many plant-pathogenic bacteria and fungi deploy effector proteins that down-regulate plant defense responses and reprogram plant metabolism for colonization and survival in planta. Kiwellin (KWL) proteins are a widespread family of plant-defense proteins that target these microbial effectors. The KWL1 protein from maize (corn, Zea mays) specifically inhibits the enzymatic activity of the secreted chorismate mutase Cmu1, a virulence-promoting effector of the smut fungus Ustilago maydis. In addition to KWL1, 19 additional KWL paralogs have been identified in maize. Here, we investigated the structure and mechanism of the closest KWL1 homolog, KWL1-b (ZEAMA_GRMZM2G305329). We solved the Cmu1–KWL1-b complex to 2.75 Å resolution, revealing a highly symmetric Cmu1–KWL1-b heterotetramer in which each KWL1-b monomer interacts with a monomer of the Cmu1 homodimer. The structure also revealed that the overall architecture of the heterotetramer is highly similar to that of the previously reported Cmu1–KWL1 complex. We found that upon U. maydis infection of Z. mays, KWL1-b is expressed at significantly lower levels than KWL1 and exhibits differential tissue-specific expression patterns. We also show that KWL1-b inhibits Cmu1 activity similarly to KWL1. We conclude that KWL1 and KWL1-b are part of a redundant defense system that tissue-specifically targets Cmu1. This notion was supported by the observation that both KWL proteins are carbohydrate-binding proteins with distinct and likely tissue-related specificities. Moreover, binding by Cmu1 modulated the carbohydrate-binding properties of both KWLs. These findings indicate that KWL proteins are part of a spatiotemporally coordinated, plant-wide defense response comprising proteins with overlapping activities.

The basidiomycete Ustilago maydis causes smut disease in maize, causing substantial losses in world corn production. This nonobligate pathogen penetrates the plant cell wall with the help of appressoria and then establishes an extensive biotrophic interaction, where the hyphae are tightly encased by the plant plasma membrane., The biotrophic fungus Ustilago maydis harbors a chitin deacetylase (CDA) family of six active genes as well as one pseudogene which are differentially expressed during colonization. This includes one secreted soluble CDA (Cda4) and five putatively glycosylphosphatidylinositol (GPI)-anchored CDAs, of which Cda7 belongs to a new class of fungal CDAs. Here, we provide a comprehensive functional study of the entire family. While budding cells of U. maydis showed a discrete pattern of chitosan staining, biotrophic hyphae appeared surrounded by a chitosan layer. We purified all six active CDAs and show their activity on different chitin substrates. Single as well as multiple cda mutants were generated and revealed a virulence defect for mutants lacking cda7. We implicated cda4 in production of the chitosan layer surrounding biotrophic hyphae and demonstrated that the loss of this layer does not reduce virulence. By combining different cda mutations, we detected redundancy as well as specific functions for certain CDAs. Specifically, certain combinations of mutations significantly affected virulence concomitantly with reduced adherence, appressorium formation, penetration, and activation of plant defenses. Attempts to inactivate all seven cda genes simultaneously were unsuccessful, and induced depletion of cda2 in a background lacking the other six cda genes illustrated an essential role of chitosan for cell wall integrity.

The tight interaction between pathogens and their hosts results in reciprocal selective forces that impact the genetic diversity of the interacting species. The footprints of this selection differ between pathosystems because of distinct life-history traits, demographic histories, or genome architectures. Here, we studied the genome-wide patterns of genetic diversity of 22 isolates of the causative agent of the corn smut disease, Ustilago maydis, originating from five locations in Mexico, the presumed center of origin of this species. In this species, many genes encoding secreted effector proteins reside in so-called virulence clusters in the genome, an arrangement that is so far not found in other filamentous plant pathogens. Using a combination of population genomic statistical analyses, we assessed the geographical, historical and genome-wide variation of genetic diversity in this fungal pathogen.We report evidence of two partially admixed subpopulations that are only loosely associated with geographic origin. Using the multiple sequentially Markov coalescent model, we inferred the demographic history of the two pathogen subpopulations over the last 0.5 million years. We show that both populations experienced a recent strong bottleneck starting around 10,000 years ago, coinciding with the assumed time of maize domestication. While the genome average genetic diversity is low compared to other fungal pathogens, we estimated that the rate of non-synonymous adaptive substitutions is three times higher in genes located within virulence clusters compared to non-clustered genes, including non-clustered effector genes. These results highlight the role that these singular genomic regions play in the evolution of this pathogen.Significance statementThe maize pathogen Ustilago maydis is a model species to study fungal cell biology and biotrophic host-pathogen interactions. Population genetic studies of this species, however, were so far restricted to using a few molecular markers, and genome-wide comparisons involved species that diverged more than 20 million years ago. Here, we sequenced the genomes of 22 Mexican U. maydis isolates to study the recent evolutionary history of this species. We identified two co-existing populations that went through a recent bottleneck and whose divergence date overlaps with the time of maize domestication. Contrasting the patterns of genetic diversity in different categories of genes, we further showed that effector genes in virulence clusters display a high rate of adaptive mutations, highlighting the importance of these effector arrangements for the adaptation of U. maydis to its host.

Due to their biotechnological relevance as well as their importance as disease agents, filamentous fungi and oomycetes have been prime candidates for genetic selection and in vitro manipulation for decades. With the advent of new genome editing technologies such manipulations have reached a new level of speed and sophistication. The CRISPR-Cas9 genome editing technology in particular has revolutionized the ways how desired mutations can be introduced. To date, the CRISPR-Cas9 genome editing system has been established in more than 40 different species of filamentous fungi and oomycetes. In this review we describe the various approaches taken to assure expression of the components necessary for editing and describe the varying strategies used to achieve gene disruptions, gene replacements and precise editing. We discuss potential problems faced when establishing the system, propose ways to circumvent them and suggest future approaches not yet realized in filamentous fungi or oomycetes.

Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses1,2. The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway3. Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.

The biotrophic basidiomycete fungus Ustilago maydis causes smut disease in maize. Hallmarks of the disease are characteristic large tumors in which dark pigmented spores are formed. Here, we functionally characterized a novel core effector lep1 (late effector protein 1) which is highly expressed during tumor formation and contributes to virulence. We characterize lep1 mutants, localize the protein, determine phenotypic consequences upon deletion as well as constitutive expression, and analyze relationships with the repellent protein Rep1 and hydrophobins. In tumors, lep1 mutants show attenuated hyphal aggregation, fail to undergo massive late proliferation and produce only a few spores. Upon constitutive expression, cell aggregation is induced and the surface of filamentous colonies displays enhanced hydrophobicity. Lep1 is bound to the cell wall of biotrophic hyphae and associates with Rep1 when constitutively expressed in hyphae. We conclude that Lep1 acts as a novel kind of cell adhesin which functions together with other surface-active proteins to allow proliferation of diploid hyphae as well as for induction of the morphological changes associated with spore formation.

The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals., The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.

During establishment of arbuscular mycorrhizal symbioses, fungal hyphae invade root cells producing transient tree-like structures, the arbuscules, where exchange of photosynthates for soil minerals occurs. Arbuscule formation and collapse lead to rapid production and degradation of plant and fungal membranes, their spatiotemporal dynamics directly influencing nutrient exchange. We determined the ultra-structural details of both membrane surfaces and the interstitial apoplastic matrix by transmission electron microscopy tomography during growth and senescence of Rhizophagus irregularis arbuscules in rice. Invasive growth of arbuscular hyphae was associated with abundant fungal membrane tubules (memtubs) and plant peri-arbuscular membrane evaginations. Similarly, the phylogenetically distant arbuscular mycorrhizal fungus, Gigaspora rosea, and the fungal maize pathogen, Ustilago maydis, developed memtubs while invading host cells, revealing structural commonalities independent of the mutualistic or parasitic outcome of the interaction. Additionally, extracellular vesicles formed continuously in the peri-arbuscular interface from arbuscule biogenesis to senescence, suggesting an involvement in inter-organismic signal and nutrient exchange throughout the arbuscule lifespan. The symbiosis of mycorrhizal fungi and roots involves invasion of plant cells, followed by collapse of the intracellular fungal arbuscules. Ultra-structural details of this interaction show continuous formation of extracellular vesicles at the peri-arbuscular space.

In many organisms, two component systems have evolved to discriminate self from nonself. While the molecular function of the two components has been elucidated in several systems, the evolutionary events leading to the large number of different specificities for self–nonself recognition found in most systems remain obscure. We have investigated the variation within a multiallelic nonself recognition system in the phytopathogenic basidiomycete Ustilago maydis by means of sequence analysis and functional studies. The multiallelic b mating type locus of U. maydis ensures outbreeding during sexual development. Nonself recognition is specified by the two homeodomain proteins, bE and bW, encoded by the b locus. While bE–bW combinations from the same allele do not dimerize, bE and bW proteins originating from different alleles form a heterodimeric complex that functions as master regulator for sexual and pathogenic development. We show that novel specificities of the b mating type locus have arisen by single homologous recombination events between distinct b alleles that lead to a simultaneous exchange of subdomains involved in dimerization in both bE and bW, altering the specificity of both proteins in a single step.

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.

Kiwellin proteins of plants have previously been recognized as important human allergens, however their biological role remained unknown. Here, we discuss recent progress towards an understanding of their role as plant-defense proteins towards pathogenic fungi and beyond.

We would like to thank Abigail LaBella for sharing the pictures taking during the session and Felicia Wu for her suggestions. We are grateful to all the colleagues that helped leading the discussion groups and all the participants of the session.

Regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling negatively. To broaden an understanding of the roles of RGS proteins in fungal pathogens, we functionally characterized the three RGS protein-encoding genes (rgs1, rgs2 and rgs3) in the phytopathogenic fungus Ustilago maydis. It was found that RGS proteins played distinct roles in the regulation of development and virulence. rgs1 had a minor role in virulence when deleted in a solopathogenic strain. In crosses, rgs1 was dispensable for mating and filamentation, but was required for teliospore production. Haploid rgs2 mutants were affected in cell morphology, growth, mating and were unable to cause disease symptoms in crosses. However, virulence was unaffected when rgs2 was deleted in a solopathogenic strain, suggesting an exclusive involvement in pre-fusion events. These rgs2 phenotypes are likely connected to elevated intracellular cAMP levels. rgs3 mutants were severely attenuated in mating, in their response to pheromone, virulence and formation of mature teliospores. The mating defect could be traced back to reduced expression of the transcription factor rop1. It was speculated that the distinct roles of the three U. maydis RGS proteins were achieved by direct modulation of the Gα subunit-activated signaling pathways as well as through Gα-independent functions.

Plant-pathogenic fungi hijack their hosts by secreting effector proteins. Effectors serve to suppress plant immune responses and modulate the host metabolism to benefit the pathogen. Smut fungi are biotrophic pathogens that also parasitize important cereals, including maize1. Symptom development is usually restricted to the plant inflorescences. Ustilago maydis is an exception in its ability to cause tumours in both inflorescences and leaves of maize, and in inducing anthocyanin biosynthesis through the secreted Tin2 effector2,3. How the unique lifestyle of U. maydis has evolved remains to be elucidated. Here we show that Tin2 in U. maydis has been neofunctionalized. We functionally compared Tin2 effectors of U. maydis and the related smut Sporisorium reilianum, which results in symptoms only in the inflorescences of maize and fails to induce anthocyanin. We show that Tin2 effectors from both fungi target distinct paralogues of a maize protein kinase, leading to stabilization and inhibition, respectively. An ancestral Tin2 effector functionally replaced the virulence function of S. reilianum Tin2 but failed to induce anthocyanin, and was unable to substitute for Tin2 in U. maydis. This shows that Tin2 in U. maydis has acquired a specialized function, probably connected to the distinct pathogenic lifestyle of this fungus.

To cause disease in maize, the biotrophic fungus Ustilago maydis secretes a large arsenal of effector proteins. Here, we functionally characterize the repetitive effector Rsp3 (repetitive secreted protein 3), which shows length polymorphisms in field isolates and is highly expressed during biotrophic stages. Rsp3 is required for virulence and anthocyanin accumulation. During biotrophic growth, Rsp3 decorates the hyphal surface and interacts with at least two secreted maize DUF26-domain family proteins (designated AFP1 and AFP2). AFP1 binds mannose and displays antifungal activity against the rsp3 mutant but not against a strain constitutively expressing rsp3. Maize plants silenced for AFP1 and AFP2 partially rescue the virulence defect of rsp3 mutants, suggesting that blocking the antifungal activity of AFP1 and AFP2 by the Rsp3 effector is an important virulence function. Rsp3 orthologs are present in all sequenced smut fungi, and the ortholog from Sporisorium reilianum can complement the rsp3 mutant of U. maydis, suggesting a novel widespread fungal protection mechanism., The fungus Ustilago maydis secretes many effector proteins to cause disease in maize. Here, Ma et al. show that the repetitive effector Rsp3 is required for virulence by inhibiting the antifungal activity of two mannose-binding proteins that are secreted by the plant cells.

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 β-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.

The smut fungus Ustilago maydis is an established model organism for elucidating how biotrophic pathogens colonize plants and how gene families contribute to virulence. Here we describe a step by step protocol for the generation of CRISPR plasmids for single and multiplexed gene editing in U. maydis. Furthermore, we describe the necessary steps required for generating edited clonal populations, losing the Cas9 containing plasmid, and for selecting the desired clones.

Genome biology and evolution : GBE 10(2), 629-645 (2018). doi:10.1093/gbe/evy023, Published by Oxford Univ. Press, Oxford

Regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling negatively. To broaden an understanding of the roles of RGS proteins in fungal pathogens, we functionally characterized the three RGS protein-encoding genes (rgs1, rgs2 and rgs3) in the phytopathogenic fungus Ustilago maydis. It was found that RGS proteins played distinct roles in the regulation of development and virulence. rgs1 had a minor role in virulence when deleted in a solopathogenic strain. In crosses, rgs1 was dispensable for mating and filamentation, but was required for teliospore production. Haploid rgs2 mutants were affected in cell morphology, growth, mating and were unable to cause disease symptoms in crosses. However, virulence was unaffected when rgs2 was deleted in a solopathogenic strain, suggesting an exclusive involvement in pre-fusion events. These rgs2 phenotypes are likely connected to elevated intracellular cAMP levels. rgs3 mutants were severely attenuated in mating, in their response to pheromone, virulence and formation of mature teliospores. The mating defect could be traced back to reduced expression of the transcription factor rop1. It was speculated that the distinct roles of the three U. maydis RGS proteins were achieved by direct modulation of the Gα subunit-activated signaling pathways as well as through Gα-independent functions.

Biotrophic fungal plant pathogens establish an intimate relationship with their host to support the infection process. Central to this strategy is the secretion of a range of protein effectors that enable the pathogen to evade plant immune defences and modulate host metabolism to meet its needs. In this Review, using the smut fungus Ustilago maydis as an example, we discuss new insights into the effector repertoire of smut fungi that have been gained from comparative genomics and discuss the molecular mechanisms by which U. maydis effectors change processes in the plant host. Finally, we examine how the expression of effector genes and effector secretion are coordinated with fungal development in the host.

The interaction of microbes with “signature” plants is largely governed by secreted effector proteins, which serve to dampen plant defense responses and modulate host cell processes. Secreted effectors can function either in the apoplast or within plant cell compartments. How oomycetes and fungi translocate their effectors to plant cells is still poorly understood and controversial. While most oomycete effectors share a common ‘signature’ that was proposed to mediate their uptake via endocytosis, fungal effectors display no conserved motifs at the primary amino acid sequence level. Here we summarize current knowledge in the field of oomycete and fungal effector uptake and highlight emerging themes that may unite rather than set apart these unrelated filamentous pathogens.

Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake. The assay exploits the ability of the bacterial biotin ligase BirA to biotinylate any protein that carries a short peptide (Avitag). It is based on the stable expression of BirA in the cytoplasm of maize plants and on engineering of Ustilago maydis strains to secrete Avitagged effectors. We demonstrate translocation of a number of effectors in the U. maydis-maize system and show data that suggest that the uptake mechanism could be rather nonspecific The assay promises to be a powerful tool for the classification of effectors as well as for the functional study of effector uptake mechanism not only in the chosen system but more generally for systems where biotrophic interactions are established.

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

In the ten years since the genome sequence of the basidiomycete corn smut fungus Ustilago maydis was published, additional genomes of smut species infecting different hosts became available. In addition, the genomes of related Malassezia species causing skin diseases and of Pseudozyma species not known to infect plants were determined. As secreted proteins are critical virulence determinants in U. maydis we compare here the secretomes of 12 basidiomycete species to gain information about their composition and conservation. For this we classify secreted proteins into those with and without domains using InterPro scans. Homology among proteins is inferred by building clusters based on pairwise similarities and cluster presence is then assessed in the different species. We detect in particular a strong correspondence between the secretomes of Pseudozyma species and plant infecting smuts. Furthermore, we identify a high proportion of secreted proteins to be part of gene families and present an advancement of the CRISPR-Cas9 technology for simultaneous disruption of multiple genes in U. maydis using five genes of the eff1 family as example.

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. DOI: http://dx.doi.org/10.7554/eLife.20522.001, eLife digest Fungi cause many diseases in plants, and reduce the yield of important crops like wheat, corn and rice – all of which belong to the family of grasses. Much research into how disease-causing fungi infect plants will look at a given fungus that infects a specific plant in order to understand plant diseases in general. Over the years, scientists have generated suites of research tools to study these pairs of fungi and plants. However, many of these organism pairs (often called “model pathosystems”) have drawbacks when it comes to research in the laboratory, either on the side of the fungus or the side of plant. Brachypodium is a small grass that grows quickly and, unlike crop plants, it grows well in the laboratory. These characteristics make Brachypodium a promising model organism for studying many aspects of plant biology. Recently, a fungus called Ustilago bromivora – which is related to a fungus that infects corn – was reported to infect Brachypodium. This raised the question: could this fungus and this small grass become a new model pathosystem? Rabe, Bosch et al. set out to answer this question and now provide a toolkit that will help to establish U. bromivora and Brachypodium as a new model pathosystem. In all of U. bromivora’s close relatives, two compatible strains must meet and mate before the fungus can infect the plant; first Rabe, Bosch et al. confirmed that this is also the case for U. bromivora. Studying the life cycle of the U. bromivora fungus also unexpectedly revealed that while both mating partners are needed to infect the plant, only one of the strains survives outside of the plant after the infection. This phenomenon, referred to as a “mating type bias”, has been described for a few other related fungi. Next, Rabe, Bosch et al. conducted a genetic screen and identified two compatible strains that can grow without the plant as yeast-like cells. This means that these cells can be manipulated genetically, and indeed protocols to grow and genetically engineer the fungus and plant to address different research questions are included in the toolkit as well. Other new tools include the complete genetic sequence of the fungus with all its genes annotated, and a dataset of which genes are active in U. bromivora growing yeast-like in liquid culture versus those active when the fungus grows as a pathogen inside the plant. Together these new tools and datasets will provide a foundation to study different aspects of the interactions between grasses and disease-causing fungi. This in turn may lead to new methods to reduce fungal growth and reduce yield losses caused by fungal diseases in crop plants. Finally, the discovery that U. bromivora shows a mating type bias could provide a starting point for future studies into sexual reproduction in fungi and how new species arise. DOI: http://dx.doi.org/10.7554/eLife.20522.002

Genome biology and evolution 8(3), 681 - 704 (2016). doi:10.1093/gbe/evw026, Published by Oxford Univ. Press, Oxford

The maize pathogenic fungus Ustilago maydis experiences endoplasmic reticulum (ER) stress during plant colonization and relies on the unfolded protein response (UPR) to cope with this stress. We identified the U. maydis co-chaperone, designated Dnj1, as part of this conserved cellular response to ER stress. ∆dnj1 cells are sensitive to the ER stressor tunicamycin and display a severe virulence defect in maize infection assays. A dnj1 mutant allele unable to stimulate the ATPase activity of chaperones phenocopies the null allele. A Dnj1-mCherry fusion protein localizes in the ER and interacts with the luminal chaperone Bip1. The Fusarium oxysporum Dnj1 ortholog contributes to the virulence of this fungal pathogen in tomato plants. Unlike the human ortholog, F. oxysporum Dnj1 partially rescues the virulence defect of the Ustilago dnj1 mutant. By enabling the fungus to restore ER homeostasis and maintain a high secretory activity, Dnj1 contributes to the establishment of a compatible interaction with the host. Dnj1 orthologs are present in many filamentous fungi, but are absent in budding and fission yeasts. We postulate a conserved and essential role during virulence for this class of co-chaperones.

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.

The biotrophic basidiomycete fungus Ustilago maydis causes smut disease in maize. Hallmarks of the disease are large tumors that develop on all aerial parts of the host in which dark pigmented teliospores are formed. We have identified a member of the WOPR family of transcription factors, Ros1, as major regulator of spore formation in U. maydis. ros1 expression is induced only late during infection and hence Ros1 is neither involved in plant colonization of dikaryotic fungal hyphae nor in plant tumor formation. However, during late stages of infection Ros1 is essential for fungal karyogamy, massive proliferation of diploid fungal cells and spore formation. Premature expression of ros1 revealed that Ros1 counteracts the b-dependent filamentation program and induces morphological alterations resembling the early steps of sporogenesis. Transcriptional profiling and ChIP-seq analyses uncovered that Ros1 remodels expression of about 30% of all U. maydis genes with 40% of these being direct targets. In total the expression of 80 transcription factor genes is controlled by Ros1. Four of the upregulated transcription factor genes were deleted and two of the mutants were affected in spore development. A large number of b-dependent genes were differentially regulated by Ros1, suggesting substantial changes in this regulatory cascade that controls filamentation and pathogenic development. Interestingly, 128 genes encoding secreted effectors involved in the establishment of biotrophic development were downregulated by Ros1 while a set of 70 “late effectors” was upregulated. These results indicate that Ros1 is a master regulator of late development in U. maydis and show that the biotrophic interaction during sporogenesis involves a drastic shift in expression of the fungal effectome including the downregulation of effectors that are essential during early stages of infection., Author Summary The fungus Ustilago maydis is a pathogen of maize which induces tumor formation in the infected tissue. In these tumors huge amounts of fungal spores develop. As a biotrophic pathogen, U. maydis establishes itself in the plant with the help of a large number of secreted effector proteins. Many effector proteins are important for virulence because they counteract plant defense reactions. In this manuscript we have identified and characterized Ros1, a master regulator for the late stages of U. maydis development. This transcription factor is expressed late during infection and controls nuclear fusion, hyphal aggregation and late proliferation. ros1 mutants are still able to induce tumor formation but these are a dead end because they do not contain any spores. We show that Ros1 interferes with the early regulatory cascade controlled by a complex of two homeodomain proteins. In addition, Ros1 triggers a major switch in the effector repertoire, suggesting that different sets of effectors are needed for different stages of fungal development inside the plant.