Conservation of the Ustilago maydis effector See1 in related smuts

Ustilago maydis is a biotrophic fungus that induces formation of tumors in maize (Zea mays L). In a recent study we identified See1 (Seedling efficient effector 1) as an U. maydis organ-specific effector required for tumor formation in leaves. See1 is required for U. maydis induced reactivation of plant DNA synthesis during leaf tumor progression. The protein is secreted from biotrophic hyphae and localizes to the cytoplasm and nucleus of plant cell. See1 interacts with maize SGT1, a cell cycle and immune regulator, interfering with its MAPK-triggered phosphorylation. Here, we present new data on the conservation of See1 in other closely related smuts and experimental data on the functionality of See1 ortholog in Ustilago hordei, the causal agent of barley covered smut disease.     

Genetic and biochemical insights into the itaconate pathway of Ustilago maydis enable enhanced production

The Ustilaginaceae family of smut fungi, especially Ustilago maydis, gained biotechnological interest over the last years, amongst others due to its ability to naturally produce the versatile bio-based building block itaconate. Along with itaconate, U. maydis also produces 2-hydroxyparaconate. The latter was proposed to be derived from itaconate, but the underlying biochemistry and associated genes were thus far unknown. Here, we confirm that 2-hydroxyparaconate is a secondary metabolite of U. maydis and propose an extension of U. maydis’ itaconate pathway from itaconate to 2-hydroxyparaconate. This conversion is catalyzed by the P450 monooxygenase Cyp3, encoded by cyp3, a gene, which is adjacent to the itaconate gene cluster of U. maydis. By deletion of cyp3 and simultaneous overexpression of the gene cluster regulator ria1, it was possible to generate an itaconate hyper producer strain, which produced up to 4.5–fold more itaconate in comparison to the wildtype without the by-product 2-hydroxyparaconate. By adjusting culture conditions in controlled pulsed fed-batch fermentations, a product to substrate yield of 67% of the theoretical maximum was achieved. In all, the titer, rate and yield of itaconate produced by U. maydis was considerably increased, thus contributing to the industrial application of this unicellular fungus for the biotechnological production of this valuable biomass derived chemical.     

Pep1, a Secreted Effector Protein of Ustilago maydis,Is Required for Successful Invasion of Plant Cells

The basidiomycete Ustilago maydis causes smut disease in maize. Colonization of the host plant is initiated by direct penetration of cuticle and cell wall of maize epidermis cells. The invading hyphae are surrounded by the plant plasma membrane and proliferate within the plant tissue. We identified a novel secreted protein, termed Pep1, that is essential for penetration. Disruption mutants of pep1 are not affected in saprophytic growth and develop normal infection structures. However, Δpep1 mutants arrest during penetration of the epidermal cell and elicit a strong plant defense response. Using Affymetrix maize arrays, we identified 116 plant genes which are differentially regulated in Δpep1 compared to wild type infections. Most of these genes are related to plant defense. By in vivo immunolocalization, live-cell imaging and plasmolysis approaches, we detected Pep1 in the apoplastic space as well as its accumulation at sites of cell-to-cell passages. Site-directed mutagenesis identified two of the four cysteine residues in Pep1 as essential for function, suggesting that the formation of disulfide bridges is crucial for proper protein folding. The barley covered smut fungus Ustilago hordei contains an ortholog of pep1 which is needed for penetration of barley and which is able to complement the U. maydis Δpep1 mutant. Based on these results, we conclude that Pep1 has a conserved function essential for establishing compatibility that is not restricted to the U. maydis / maize interaction.     

Transgenic maize plants expressing the Totivirus antifungal protein, KP4, are highly resistant to corn smut

The corn smut fungus, Ustilago maydis, is a global pathogen responsible for extensive agricultural losses. Control of corn smut using traditional breeding has met with limited success because natural resistance to U. maydis is organ specific and involves numerous maize genes. Here, we present a transgenic approach by constitutively expressing the Totivirus antifungal protein KP4, in maize. Transgenic maize plants expressed high levels of KP4 with no apparent negative impact on plant development and displayed robust resistance to U. maydis challenges to both the stem and ear tissues in the greenhouse. More broadly, these results demonstrate that a high level of organ independent fungal resistance can be afforded by transgenic expression of this family of antifungal proteins.     

Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungusUstilago maydis

Pathogenic development ofUstilago maydis, the causative agent of corn smut disease, is a multistep process. Compatible yeast-like cells fuse and this generates the infectious dikaryon which grows filamentously. Having entered the plant the dikaryon induces tumors in its host in which massive proliferation of fungal material, karyogamy and spore formation occur. In order to follow fungal development from the initial steps to the final stage we have expressed the green fluorescent protein (GFP) fromAequorea victoria as a vital marker inU. maydis and demonstrate that GFP-tagged strains can be used to study host-pathogen interactions in vivo.     

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.     

The Influence of Ustilago maydis Upon Free Radicals Concentration in the Reproduction Organs of Maize

The reproduction organs of maize are those most often attacked by Ustilago maydis. In the male organs it produces a decline in free radicals concentration and, conversely, a rise of concentration in the female organs (in ears). The smut proper, grown on the flower, exhibits an essentially higher free radicals concentration than the smut grown out on the ear of that plant. A smut mixture from flower and ear has only a slightly higher free radicals concentration than the separate ear smut, but an essentially lower one than the smut of the flower.     

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.     

Isolation of the ERG2 Gene,Encoding Δ8 → Δ7 Sterol Isomerase,from the Maize Smut Pathogen Ustilago maydis

Bailey, A. M., Burden, R. S., James, C. S., Keon, J. P. R., Croxen, R., Bard, M., and Hargreaves, J. A. 1993. Isolation of the ERG2 gene, encoding Δ⁸ → Δ⁷ sterol isomerase, from the maise smut pathogen Ustilago maydis. Experimental Mycology 18, 87-92. The ERG2 gene encoding Δ⁸ → Δ⁷ sterol isomerase has been isolated from the fungal plant pathogen Ustilago maydis. This was accomplished by screening an U. maydis genomic library with a fragment of the Saccharomyces cerevisiae ERG2 gene. The identity of the U. maydis ERG2 gene was confirmed by complementation of an U. maydis Erg2 mutant and by comparing the deduced amino acid sequence encoded by the U. maydis ERG2 gene with that of the S. cerevisiae ERG2 gene product.     

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.     

Flamingomyces and Parvulago, new genera of marine smut fungi (Ustilaginomycotina)

Teliospore walls, teliospore germinations, hyphal septations, cellular interactions, and nucleotide sequences from the D1/D2 region of the nuLSU rRNA gene of the marine smut fungi Melanotaenium ruppiae and Ustilago marina were examined and compared with findings in other Ustilaginomycotina. The data show that Melanotaenium ruppiae belongs to the Urocystaceae and Ustilago marina to the Ustilaginaceae. Within the Urocystaceae, Melanotaenium ruppiae is morphologically similar to Melanustilospora and Vankya. However, according to the molecular results Melanotaenium ruppiae can neither be ascribed to Melanustilospora nor to Vankya. Therefore, the new genus Flamingomyces is proposed for Melanotaenium ruppiae. Ustilago marina differs from the other Ustilaginaceae in the mode of sporulation, which exclusively occurs at the base of the host plant culms. Accordingly, the new genus Parvulago is proposed for Ustilago marina.     

Metamorphosis of the Basidiomycota Ustilago maydis: Transformation of yeast-like cells into basidiocarps

Ustilago maydis (DC) Cda., a phytopathogenic Basidiomycota, is the causal agent of corn smut. During its life cycle U. maydis alternates between a yeast-like, haploid nonpathogenic stage, and a filamentous, dikaryotic pathogenic form that invades the plant and induces tumor formation. As all the members of the Subphylum Ustilaginomycotina, U. maydis is unable to form basidiocarps, instead it produces teliospores within the tumors that germinate forming a septate basidium (phragmobasidium). We have now established conditions allowing a completely different developmental program of U. maydis when grown on solid medium containing auxins in dual cultures with maize embryogenic calli. Under these conditions U. maydis forms large hemi-spheroidal structures with all the morphological and structural characteristics of gastroid-type basidiocarps. These basidiocarps are made of three distinct hyphal layers, the most internal of which (hymenium) contains non-septate basidia (holobasidia) from which four basidiospores develop. In basidiocarps meiosis and genetic recombination occur, and meiotic products (basidiospores) segregate in a Mendelian fashion. These results are evidence of sexual cycle completion of an Ustilaginomycotina in vitro, and the demonstration that, besides its quasi-obligate biotrophic pathogenic mode of life, U. maydis possesses the genetic program to form basidiocarps as occurs in saprophytic Basidiomycota species.     

Pheromone-induced G2 arrest in the phytopathogenic fungus Ustilago maydis

In the corn smut fungus Ustilago maydis, pathogenic development is initiated when two compatible haploid cells fuse and form the infectious dikaryon. Mating is dependent on pheromone recognition by compatible cells. In this report, we set out to evaluate the relationship between the cell cycle and the pheromone response in U. maydis. To achieve this, we designed a haploid pheromone-responsive strain that is able to faithfully reproduce the native mating response in nutrient-rich medium. Addition of synthetic pheromone to the responsive strain induces the formation of mating structures, and this response is abolished by mutations in genes encoding components of the pheromone signal transduction cascade. After recognition of pheromone, U. maydis cells arrest the cell cycle in a postreplicative stage. Visualization of the nucleus and microtubule organization indicates that the arrest takes place at the G₂ phase. Chemical-induced cell cycle arrest and release in the presence of pheromone further support this conclusion.     

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.     

Development and validation of microsatellite markers for Karnal bunt (Tilletia indica) and loose smut (Ustilago segetum tritici) of wheat from related fungal species

Simple sequence repeats (SSRs) are preferred molecular markers because of their abundance, robustness, high reproducibility, high efficiency in detecting variation and suitability for high‐throughput analysis. In this study, an attempt was made to mine and analyse the SSRs from the genomes of two seed‐borne fungal pathogens, viz Ustilago maydis, which causes common smut of maize, and Tilletia horrida, the cause of rice kernel smut. After elimination of redundant sequences, 2,703 SSR loci of U. maydis were identified. Of the remaining SSRS, 44.5% accounted for di‐nucleotide repeats followed by 29.8% and 2.7% tri‐ and tetranucleotide repeats, respectively. Similarly, 2,638 SSR loci were identified in T. horrida, of which 20.2% were di‐nucleotide, 50.4% tri‐ and 20.5% tetra‐nucleotide repeats. A set of 65 SSRs designed from each fungus were validated, which yielded 23 polymorphic SSRs from Ustilago and 21 from Tilletia. These polymorphic SSR loci were also successfully cross‐amplified with the Ustilago segetum tritici and Tilletia indica. Principal coordinate analysis of SSR data clustered isolates according to their respective species. These newly developed and validated microsatellite markers may have immediate applications for detection of genetic variability and in population studies of bunt and smut of wheat and other related host plants. Moreover, this is first comprehensive report on molecular markers suitable for variability studies in wheat seed‐borne pathogens.     

玉米瘤黑粉菌的寄生策略及其调控机制

玉米瘤黑粉病是由担子菌Ustilago maydis对玉米的活体寄生所引起的真菌病害。该病原菌为双相型真菌,需要寄生于玉米植株来完成其有性生殖过程。综合相关研究报道,本文把U.maydis对寄主植物的寄生过程划分为7个阶段,包括形成致病性双核菌丝体、附着寄主植物表面、穿透寄主表皮、消减寄主防御反应、在寄主体内菌丝增殖、使寄主瘤变和生成厚垣孢子等。围绕寄生进程特点和关键基因,分别阐述了各个阶段的相关调控机制以及对寄主植物的致病性;展现了U.maydis为达到有性生殖目的而实施步步为营的寄生策略。本文对U.maydis寄生过程的阶段划分,有助于人们深入了解U.maydis与寄主植物之间互作机制、提供相关病害防控新思路。