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.

My Personal Journey from the Fascination for Phages to a Tumor-Inducing Fungal Pathogen of Corn.

My path in science began with a fascination for microbiology and phages and later involved a switch of subjects to the fungus Ustilago maydis and how it causes disease in maize. I will not provide a review of my work but rather focus on decisive findings, serendipitous, lucky moments when major advances made the U. maydis-maize system what it is now-a well-established model for biotrophic fungi. I also want to share with you the joy of finding the needle in a haystack at the very end of my scientific career, a fungal structure likely used for effector delivery, and how we were able to translate this into a potential application in agriculture.

A cell surface-exposed protein complex with an essential virulence function in Ustilago maydis.

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.

A small Ustilago maydis effector acts as a novel adhesin for hyphal aggregation in plant tumors.

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.

Population Genomics of the Maize Pathogen Ustilago maydis: Demographic History and Role of Virulence Clusters in Adaptation.

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 Myr. We show that both populations experienced a recent strong bottleneck starting around 10,000 years ago, coinciding with the assumed time of maize domestication. Although the genome average genetic diversity is low compared with other fungal pathogens, we estimated that the rate of nonsynonymous adaptive substitutions is three times higher in genes located within virulence clusters compared with nonclustered genes, including nonclustered effector genes. These results highlight the role that these singular genomic regions play in the evolution of this pathogen.

Nuclear status and leaf tumor formation in the Ustilago maydis-maize pathosystem.

Ustilago maydis is a biotrophic fungus causing smut disease in corn. The infectious forms are dikaryotic hyphae. Here we analyze mutants lacking the nlt1 transcription factor and investigate why these mutants are unable to induce leaf tumors. The study involved reverse genetics, complementation, epistasis analysis, microscopy, gene expression analysis by quantitative reverse transcriptase PCR and virulence assays. We show that nlt1 mutants colonize maize leaves efficiently but fail to undergo karyogamy and are attenuated in late proliferation. Nlt1 activates transcription of ros1, a transcription factor controlling karyogamy, and represses see1, an effector previously shown to contribute to leaf tumor induction. In mononuclate solopathogenic strains, nlt1 mutants cause attenuated leaf tumor formation. In actively dividing maize organs, nlt1 mutants undergo karyogamy and induce tumor formation. Sporisorium reilianum, a smut fungus unable to induce leaf tumors, possesses an ortholog of nlt1 that controls the fusion of dikaryotic nuclei late in infection during cob colonization. Our results have established a regulatory connection between nlt1, ros1 and see1 and suggest the existence of two stages contributing to leaf tumor formation, one before nuclear fusion and involving nlt1 and one after karyogamy that is nlt1 independent.

Chitosan and Chitin Deacetylase Activity Are Necessary for Development and Virulence of Ustilago maydis.

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.IMPORTANCE 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. For successful invasion and development in plant tissue, recognition of conserved fungal cell wall components such as chitin by the plant immune system needs to be avoided or suppressed. One strategy to achieve this lies in the modification of chitin to chitosan by chitin deacetylases (CDAs). U. maydis has seven cda genes. This study reveals discrete as well as redundant contributions of these genes to virulence as well as to cell wall integrity. Unexpectedly, the inactivation of all seven genes is not tolerated, revealing an essential role of chitosan for viability.

Cell wall-associated effectors of plant-colonizing fungi.

Plant-colonizing fungi secrete a cocktail of effector proteins during colonization. After secretion, some of these effectors are delivered into plant cells to directly dampen the plant immune system or redirect host processes benefitting fungal growth. Other effectors function in the apoplastic space either as released proteins modulating the activity of plant enzymes associated with plant defense or as proteins bound to the fungal cell wall. For such fungal cell wall-bound effectors, we know particularly little about their molecular function. In this review, we describe effectors that are associated with the fungal cell wall and discuss how they contribute to colonization.

The insertion of a mitochondrial selfish element into the nuclear genome and its consequences.

Homing endonucleases (HE) are enzymes capable of cutting DNA at highly specific target sequences, the repair of the generated double-strand break resulting in the insertion of the HE-encoding gene ("homing" mechanism). HEs are present in all three domains of life and viruses; in eukaryotes, they are mostly found in the genomes of mitochondria and chloroplasts, as well as nuclear ribosomal RNAs. We here report the case of a HE that accidentally integrated into a telomeric region of the nuclear genome of the fungal maize pathogen Ustilago maydis. We show that the gene has a mitochondrial origin, but its original copy is absent from the U. maydis mitochondrial genome, suggesting a subsequent loss or a horizontal transfer from a different species. The telomeric HE underwent mutations in its active site and lost its original start codon. A potential other start codon was retained downstream, but we did not detect any significant transcription of the newly created open reading frame, suggesting that the inserted gene is not functional. Besides, the insertion site is located in a putative RecQ helicase gene, truncating the C-terminal domain of the protein. The truncated helicase is expressed during infection of the host, together with other homologous telomeric helicases. This unusual mutational event altered two genes: The integrated HE gene subsequently lost its homing activity, while its insertion created a truncated version of an existing gene, possibly altering its function. As the insertion is absent in other field isolates, suggesting that it is recent, the U. maydis 521 reference strain offers a snapshot of this singular mutational event.

Creating novel specificities in a fungal nonself recognition system by single step homologous recombination events.

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 two paralogous kiwellin proteins KWL1 and KWL1-b from maize are structurally related and have overlapping functions in plant defense.

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.

Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture.

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.

The functionally conserved effector Sta1 is a fungal cell wall protein required for virulence in Ustilago maydis.

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, ß-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.

Manipulation of Phytohormone Pathways by Effectors of Filamentous Plant Pathogens.

Phytohormones regulate a large variety of physiological processes in plants. In addition, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are responsible for primary defense responses against abiotic and biotic stresses, while plant growth regulators, such as auxins, brassinosteroids (BRs), cytokinins (CKs), abscisic acid (ABA), and gibberellins (GAs), also contribute to plant immunity. To successfully colonize plants, filamentous pathogens like fungi and oomycetes have evolved diverse strategies to interfere with phytohormone pathways with the help of secreted effectors. These include proteins, toxins, polysaccharides as well as phytohormones or phytohormone mimics. Such pathogen effectors manipulate phytohormone pathways by directly altering hormone levels, by interfering with phytohormone biosynthesis, or by altering or blocking important components of phytohormone signaling pathways. In this review, we outline the various strategies used by filamentous phytopathogens to manipulate phytohormone pathways to cause disease.

CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes.

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.

Arbuscular cell invasion coincides with extracellular vesicles and membrane tubules.

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.

A kiwellin disarms the metabolic activity of a secreted fungal virulence factor.

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.

Neofunctionalization of the secreted Tin2 effector in the fungal pathogen Ustilago maydis.

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.

Virulence function of the Ustilago maydis sterol carrier protein 2.

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