The core effector Cce1 is required for early infection of maize by Ustilago maydis

The biotrophic pathogen Ustilago maydis, the causative agent of corn smut disease, infects one of the most important crops worldwide – Zea mays. To successfully colonize its host, U. maydis secretes proteins, known as effectors, that suppress plant defense responses and facilitate the establishment of biotrophy. In this work, we describe the U. maydis effector protein Cce1. Cce1 is essential for virulence and is upregulated during infection. Through microscopic analysis and in vitro assays, we show that Cce1 is secreted from hyphae during filamentous growth of the fungus. Strikingly, Δcce1 mutants are blocked at early stages of infection and induce callose deposition as a plant defense response. Cce1 is highly conserved among smut fungi and the Ustilago bromivora ortholog complemented the virulence defect of the SG200Δcce1 deletion strain. These data indicate that Cce1 is a core effector with apoplastic localization that is essential for U. maydis to infect its host.     

The Transition from a Phytopathogenic Smut Ancestor to an Anamorphic Biocontrol Agent Deciphered by Comparative Whole-Genome Analysis

Pseudozyma flocculosa is related to the model plant pathogen Ustilago maydis yet is not a phytopathogen but rather a biocontrol agent of powdery mildews; this relationship makes it unique for the study of the evolution of plant pathogenicity factors. The P. flocculosa genome of ∼23 Mb includes 6877 predicted protein coding genes. Genome features, including hallmarks of pathogenicity, are very similar in P. flocculosa and U. maydis, Sporisorium reilianum, and Ustilago hordei. Furthermore, P. flocculosa, a strict anamorph, revealed conserved and seemingly intact mating-type and meiosis loci typical of Ustilaginales. By contrast, we observed the loss of a specific subset of candidate secreted effector proteins reported to influence virulence in U. maydis as the singular divergence that could explain its nonpathogenic nature. These results suggest that P. flocculosa could have once been a virulent smut fungus that lost the specific effectors necessary for host compatibility. Interestingly, the biocontrol agent appears to have acquired genes encoding secreted proteins not found in the compared Ustilaginales, including necrosis-inducing-Phytophthora-protein- and Lysin-motif- containing proteins believed to have direct relevance to its lifestyle. The genome sequence should contribute to new insights into the subtle genetic differences that can lead to drastic changes in fungal pathogen lifestyles.     

Induction of apoptosis‐like cell death and clearance of stress‐induced intracellular protein aggregates: dual roles for Ustilago maydis metacaspase Mca1

Metacaspases primarily associate with induction and execution of programmed cell death in protozoa, fungi and plants. In the recent past, several studies have also demonstrated cellular functions of metacaspases other than cell death in different organisms including yeast and protozoa. This study shows similar dual function for the only metacaspase of a biotrophic phytopathogen, Ustilago maydis. In addition to a conventional role in the induction of cell death, Mca1 has been demonstrated to play a key role in maintaining the quality of the cellular proteome. On one hand, Mca1 could be shown to bring about apoptosis‐like phenotypic changes in U. maydis on exposure to oxidative stress, on the other hand, the protein was found to regulate cellular protein quality control. U. maydis metacaspase has been found to remain closely associated with the insoluble intracellular protein aggregates, generated during an event of stress exposure to the fungus. The study, therefore, provides direct evidence for a role of U. maydis metacaspase in the clearance of the stress‐induced intracellular insoluble protein aggregates. Furthermore, host infection assays with mca1 deletion strain also revealed a role of the protein in the virulence of the fungus.     

Sporisorium reilianum possesses a pool of effector proteins that modulate virulence on maize

The biotrophic maize head smut fungus Sporisorium reilianum is a close relative of the tumour-inducing maize smut fungus Ustilago maydis with a distinct disease aetiology. Maize infection with S. reilianum occurs at the seedling stage, but spores first form in inflorescences after a long endophytic growth phase. To identify S. reilianum-specific virulence effectors, we defined two gene sets by genome comparison with U. maydis and with the barley smut fungus Ustilago hordei. We tested virulence function by individual and cluster deletion analysis of 66 genes and by using a sensitive assay for virulence evaluation that considers both disease incidence (number of plants with a particular symptom) and disease severity (number and strength of symptoms displayed on any individual plant). Multiple deletion strains of S. reilianum lacking genes of either of the two sets (sr10057, sr10059, sr10079, sr10703, sr11815, sr14797 and clusters uni5-1, uni6-1, A1A2, A1, A2) were affected in virulence on the maize cultivar ‘Gaspe Flint’, but each of the individual gene deletions had only a modest impact on virulence. This indicates that the virulence of S. reilianum is determined by a complex repertoire of different effectors which each contribute incrementally to the aggressiveness of the pathogen.     

Pathocycles: Ustilago maydis as a model to study the relationships between cell cycle and virulence in pathogenic fungi

Activation of virulence in pathogenic fungi often involves differentiation processes that need the reset of the cell cycle and induction of a new morphogenetic program. Therefore, the fungal capability to modify its cell cycle constitutes an important determinant in carrying out a successful infection. The dimorphic fungus Ustilago maydis is the causative agent of corn smut disease and has lately become a highly attractive model in addressing fundamental questions about development in pathogenic fungi. The different morphological and genetic changes of U. maydis cells during the pathogenic process advocate an accurate control of the cell cycle in these transitions. This is why this model pathogen deserves attention as a powerful tool in analyzing the relationships between cell cycle, morphogenesis, and pathogenicity. The aim of this review is to summarize recent advances in the unveiling of cell cycle regulation in U. maydis. We also discuss the connection between cell cycle and virulence and how cell cycle control is an important downstream target in the fungus-plant interaction.     

The pep4 gene encoding proteinase A is involved in dimorphism and pathogenesis of Ustilago maydis

Vacuole proteases have important functions in different physiological processes in fungi. Taking this aspect into consideration, and as a continuation of our studies on the analysis of the proteolytic system of Ustilago maydis, a phytopathogenic member of the Basidiomycota, we have analysed the role of the pep4 gene encoding the vacuolar acid proteinase PrA in the pathogenesis and morphogenesis of the fungus. After confirmation of the location of the protease in the vacuole using fluorescent probes, we obtained deletion mutants of the gene in sexually compatible strains of U. maydis (FB1 and FB2), and analysed their phenotypes. It was observed that the yeast to mycelium dimorphic transition induced by a pH change in the medium, or the use of a fatty acid as sole carbon source, was severely reduced in Δpep4 mutants. In addition, the virulence of the mutants in maize seedlings was reduced, as revealed by the lower proportion of plants infected and the reduction in size of the tumours induced by the pathogen, when compared with wild‐type strains. All of these phenotypic alterations were reversed by complementation of the mutant strains with the wild‐type gene. These results provide evidence of the importance of the pep4 gene for the morphogenesis and virulence of U. maydis.     

Endocytosis in the plant-pathogenic fungus Ustilago maydis

Summary. Filamentous fungi are an important group of tip-growing organisms, which include numerous plant pathogens such as Magnaporthe grisea and Ustilago maydis. Despite their ecological and economical relevance, we are just beginning to unravel the importance of endocytosis in filamentous fungi. Most evidence for endocytosis in filamentous fungi is based on the use of endocytic tracer dyes that are taken up into the cell and delivered to the vacuole. Moreover, genomewide screening for candidate genes in Neurospora crassa and U. maydis confirmed the presence of most components of the endocytic machinery, indicating that endocytosis participates in filamentous growth. Indeed, it was shown that in U. maydis early endosomes cluster at sites of growth, where they support morphogenesis and polar growth, most likely via endosome-based membrane recycling. In humans, such recycling processes to the plasma membrane involve small GTPases such as Rab4. A homologue of this protein is encoded in the genome of U. maydis but is absent from the yeast Saccharomyces cerevisiae, suggesting that Rab4-mediated recycling is important for filamentous growth. Furthermore, human Rab4 regulates traffic of early endosomes along microtubules, and a similar microtubule-based transport is described for U. maydis. These observations suggest that Rab4-like GTPases might regulate endosome- and microtubule-based recycling during tip growth of filamentous fungi. Correspondence and reprints: MPI für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Federal Republic of Germany.     

14-3-3 regulates the G2/M transition in the basidiomycete Ustilago maydis

14-3-3 proteins are a family of highly conserved polypeptides that function as small adaptors that facilitate a diverse array of cellular processes by binding phosphorylated target proteins. One of these processes is the regulation of the cell cycle. Here we characterized the role of Bmh1, a 14-3-3 protein, in the cell cycle regulation of the fungus Ustilago maydis. We found that this protein is essential in U. maydis and that it has roles during the G2/M transition in this organism. The function of 14-3-3 in U. maydis seems to mirror the proposed role for this protein during Schizosaccharomyces pombe cell cycle regulation. We provided evidence that in U. maydis 14-3-3 protein binds to the mitotic regulator Cdc25. Comparison of the roles of 14-3-3 during cell cycle regulation in other fungal system let us to discuss the connections between morphogenesis, cell cycle regulation and the evolutionary role of 14-3-3 proteins in fungi.     

The O-Mannosyltransferase PMT4 Is Essential for Normal Appressorium Formation and Penetration in Ustilago maydis

In Saccharomyces cerevisiae, the PMT, KRE2/MNT1, and MNN1 mannosyltransferase protein families catalyze the steps of the O-mannosylation pathway, sequentially adding mannoses to target proteins. We have identified members of all three families and analyzed their roles in pathogenesis of the maize smut fungus Ustilago maydis. Furthermore, we have shown that PMT4, one of the three PMT family members in U. maydis, is essential for tumor formation in Zea mays. Significantly, PMT4 seems to be required only for pathogenesis and is dispensable for other aspects of the U. maydis life cycle. We subsequently show that the deletion of pmt4 results in a strong reduction in the frequency of appressorium formation, with the few appressoria that do form lacking the capacity to penetrate the plant cuticle. Our findings suggest that the O-mannosylation pathway plays a key role in the posttranslational modification of proteins involved in the pathogenic development of U. maydis. The fact that PMT homologs are not found in plants may open new avenues for the development of fungal control strategies. Moreover, the discovery of a highly specific requirement for a single O-mannosyltransferase should aid in the identification of the proteins directly involved in fungal plant penetration, thus leading to a better understanding of plant–fungi interactions.     

A PCR-based system for highly efficient generation of gene replacement mutants in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

Ustilago maydis, the causative agent of corn smut disease, is one of the most versatile model systems for the study of plant pathogenic fungi. With the availability of the complete genomic sequence there is an increasing need to improve techniques for the generation of deletion mutants in order to elucidate the functions of unknown genes. Here a method is presented which allows one to generate constructs for gene replacement without the need for cloning. The 5 and 3-regions of the target gene are first amplified by PCR, and subsequently ligated directionally to a marker cassette via two distinct Sfi I sites, providing the flanking homologies needed for homologous recombination in U. maydis. Then the ligation product is used as a template for the amplification of the deletion construct, which can be used directly for transformation of U. maydis. The use of the fragments generated by PCR drastically increases the frequency of homologous recombination when compared to the linearized plasmids routinely used for gene replacement in U. maydis.Communicated by G. Jürgens     

Ustilago maydis accumulates β-carotene at levels determined by a retinal-forming carotenoid oxygenase

The basidiomycete Ustilago maydis, the causative agent of corn smut disease, has emerged as a model organism for dimorphism and fungal phytopathogenicity. In this work, we line out the key conserved enzymes for β-carotene biosynthesis encoded by the U. maydis genome and show that this biotrophic fungus accumulates β-carotene. The amount of this pigment depended on culture pH and aeration but was not affected by light and was not increased by oxidative stress. Moreover, we identified the U. maydis gene, cco1, encoding a putative β-carotene cleavage oxygenase. Heterologous overexpression and in vitro analyses of purified enzyme demonstrated that Cco1 catalyzes the symmetrical cleavage of β-carotene to yield two molecules of retinal. Analyses of β-carotene and retinal contents in U. maydis cco1 deletion and over-expression strains confirmed the enzymatic function of Cco1, and revealed that Cco1 determines the β-carotene content. Our data indicate that carotenoid biosynthesis in U. maydis is carried out to provide retinal rather than to deliver protective pigments. The U. maydis genome also encodes three potential opsins, a family of photoactive proteins that use retinal as chromophore. Two opsin genes showed different light-regulated expression patterns, suggesting specialized roles in photobiology, while no mRNA was detected for the third opsin gene in the same experiments. However, deletion of the cco1 gene, which should abolish function of all the retinal-dependent opsins, did not affect growth, morphology or pathogenicity, suggesting that retinal and opsin proteins play no relevant role in U. maydis under the tested conditions.     

A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host.     

Signaling via cAMP in fungi: interconnections with mitogen-activated protein kinase pathways

The cAMP signal transduction pathway controls a wide variety of processes in fungi. For example, considerable progress has been made in describing the involvement of cAMP pathway components in the control of morphogenesis in Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea. These morphological processes include the establishment of filamentous growth in S. cerevisiae and U. maydis, and the differentiation of an appressorial infection structure in M. grisea. The discovery that appressorium formation requires cAMP signaling provides an immediate connection to fungal virulence. This connection may have broader implications among fungal pathogens because recent work indicates that cAMP signaling controls the expression of virulence traits in the human pathogen Cryptococcus neoformans. In this fungus, cAMP also influences mating, as has been found for Schizosaccharomyces pombe and as may occur in U. maydis. Finally, cAMP and mitogen-activated protein kinase pathways appear to function coordinately to control the response of certain fungi, e.g., Saccharomyces cerevisiae and Schizosaccharomyces pombe, to environmental stress. There are clues that interconnections between these pathways may be common in the control of many fungal processes. Received: 5 June 1998 / Accepted: 11 September 1998     

Establishment of compatibility in the Ustilago maydis/maize pathosystem

The fungus Ustilago maydis is a biotrophic pathogen parasitizing on maize. The most prominent symptoms of the disease are large tumors in which fungal proliferation and spore differentiation occur. In this study, we have analyzed early and late tumor stages by confocal microscopy. We show that fungal differentiation occurs both within plant cells as well as in cavities where huge aggregates of fungal mycelium develop. U. maydis is poorly equipped with plant CWDEs and we demonstrate by array analysis that the respective genes follow distinct expression profiles at early and late stages of tumor development. For the set of three genes coding for pectinolytic enzymes, deletion mutants were generated by gene replacement. Neither single nor triple mutants were affected in pathogenic development. Based on our studies, we consider it unlikely that U. maydis feeds on carbohydrates derived from the digestion of plant cell wall material, but uses its set of plant CWDEs for softening the cell wall structure as a prerequisite for in planta growth.