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 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.

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.

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.

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.

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.

The Biotrophic Development of Ustilago maydis Studied by RNA-Seq Analysis.

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.

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 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.

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.

Three regulators of G protein signaling differentially affect mating, morphology and virulence in the smut fungus Ustilago maydis.

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.

Comparative analyses of secreted proteins in plant pathogenic smut fungi and related basidiomycetes.

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.

The WOPR Protein Ros1 Is a Master Regulator of Sporogenesis and Late Effector Gene Expression in the Maize Pathogen Ustilago maydis.

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.

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.

An assay for entry of secreted fungal effectors into plant cells.

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.

Metabolic priming by a secreted fungal effector.

Maize smut caused by the fungus Ustilago maydis is a widespread disease characterized by the development of large plant tumours. U. maydis is a biotrophic pathogen that requires living plant tissue for its development and establishes an intimate interaction zone between fungal hyphae and the plant plasma membrane. U. maydis actively suppresses plant defence responses by secreted protein effectors. Its effector repertoire comprises at least 386 genes mostly encoding proteins of unknown function and expressed exclusively during the biotrophic stage. The U. maydis secretome also contains about 150 proteins with probable roles in fungal nutrition, fungal cell wall modification and host penetration as well as proteins unlikely to act in the fungal-host interface like a chorismate mutase. Chorismate mutases are key enzymes of the shikimate pathway and catalyse the conversion of chorismate to prephenate, the precursor for tyrosine and phenylalanine synthesis. Root-knot nematodes inject a secreted chorismate mutase into plant cells likely to affect development. Here we show that the chorismate mutase Cmu1 secreted by U. maydis is a virulence factor. The enzyme is taken up by plant cells, can spread to neighbouring cells and changes the metabolic status of these cells through metabolic priming. Secreted chorismate mutases are found in many plant-associated microbes and might serve as general tools for host manipulation.

Genome editing in Ustilago maydis using the CRISPR-Cas system.

This communication describes the establishment of the type II bacterial CRISPR-Cas9 system to efficiently disrupt target genes in the fungal maize pathogen Ustilago maydis. A single step transformation of a self-replicating plasmid constitutively expressing the U. maydis codon-optimized cas9 gene and a suitable sgRNA under control of the U. maydis U6 snRNA promoter was sufficient to induce genome editing. On average 70% of the progeny of a single transformant were disrupted within the respective b gene. Without selection the self-replicating plasmid was lost rapidly allowing transient expression of the CRISPR-Cas9 system to minimize potential long-term negative effects of Cas9. This technology will be an important advance for the simultaneous disruption of functionally redundant genes and gene families to investigate their contribution to virulence of U. maydis.

Characterization of the largest effector gene cluster of Ustilago maydis.

In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.

Plant surface cues prime Ustilago maydis for biotrophic development.

Infection-related development of phytopathogenic fungi is initiated by sensing and responding to plant surface cues. This response can result in the formation of specialized infection structures, so-called appressoria. To unravel the program inducing filaments and appressoria in the biotrophic smut fungus Ustilago maydis, we exposed cells to a hydrophobic surface and the cutin monomer 16-hydroxy hexadecanoic acid. Genome-wide transcriptional profiling at the pre-penetration stage documented dramatic transcriptional changes in almost 20% of the genes. Comparisons with the U. maydis sho1 msb2 double mutant, lacking two putative sensors for plant surface cues, revealed that these plasma membrane receptors regulate a small subset of the surface cue-induced genes comprising mainly secreted proteins including potential plant cell wall degrading enzymes. Targeted gene deletion analysis ascribed a role to up-regulated GH51 and GH62 arabinofuranosidases during plant penetration. Among the sho1/msb2-dependently expressed genes were several secreted effectors that are essential for virulence. Our data also demonstrate specific effects on two transcription factors that redirect the transcriptional regulatory network towards appressorium formation and plant penetration. This shows that plant surface cues prime U. maydis for biotrophic development.

A conserved co-chaperone is required for virulence in fungal plant pathogens.

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.