Endoplasmic Reticulum Glucosidases and Protein Quality Control Factors Cooperate to Establish Biotrophy in Ustilago maydis

Secreted fungal effectors mediate plant–fungus pathogenic interactions. These proteins are typically N-glycosylated, a common posttranslational modification affecting their location and function. N-glycosylation consists of the addition, and subsequent maturation, of an oligosaccharide core in the endoplasmic reticulum (ER) and Golgi apparatus. In this article, we show that two enzymes catalyzing specific stages of this pathway in maize smut (Ustilago maydis), glucosidase I (Gls1) and glucosidase II β-subunit (Gas2), are essential for its pathogenic interaction with maize (Zea mays). Gls1 is required for the initial stages of infection following appressorium penetration, and Gas2 is required for efficient fungal spreading inside infected tissues. While U. maydis Δgls1 cells induce strong plant defense responses, Δgas2 hyphae are able to repress them, showing that slight differences in the N-glycoprotein processing can determine the extent of plant–fungus interactions. Interestingly, the calnexin protein, a central element of the ER quality control system for N-glycoproteins in eukaryotic cells, is essential for avoiding plant defense responses in cells with defective N-glycoproteins processing. Thus, N-glycoprotein maturation and this conserved checkpoint appear to play an important role in the establishment of an initial biotrophic state with the plant, which allows subsequent colonization.     

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.     

The biotechnological use and potential of plant pathogenic smut fungi

Plant pathogens of the family Ustilaginaceae parasitise mainly on grasses and cause smut disease. Among the best characterised members of this family are the covered smut fungus Ustilago hordei colonising barley and oat as well as the head smut Sporisorium reilianum and the corn smut Ustilago maydis, both infecting maize. Over the past years, U. maydis in particular has matured into a model system for diverse topics like plant–pathogen interaction, cellular transport processes or DNA repair. Consequently, a broad set of genetic, molecular and system biological methods has been established. This set currently serves as a strong foundation to improve existing and establish novel biotechnological applications. Here, we review four promising aspects covering different fields of applied science: (1) synthesis of secondary metabolites produced at fermenter level. (2) Lipases and other hydrolytic enzymes with potential roles in biocatalytic processes. (3) Degradation of ligno-cellulosic plant materials for biomass conversion. (4) Protein expression based on unconventional secretion, a novel approach inspired by basic research on mRNA transport. Thus, plant pathogenic Ustilaginaceae offer a great potential for future biotechnological applications by combining basic research and applied science.     

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 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.     

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.     

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.     

The Influence of Ustilago maydis on Free Radicals Concentration in Zea mays Tissues

In the plant Ustilago maydis causes: 1. A lowering of free radical concentrations in tissues of nodal roots and in internodial tissues close to the fungus. 2. Conversely, in tissues of the primary root, of aerial roots, of leaves and nodes 1–5, 13, 15 and 16 it causes a rise of free radicals concentration. Ustilago maydis has a similar effect upon free radicals concentration in maize leaves as Erysiphe graminis in barley leaves.     

A Secreted Effector Protein of Ustilago maydis Guides Maize Leaf Cells to Form Tumors

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.     

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 Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.¹ Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut². However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually^3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.     

Ustilago maydis Produces Cytokinins and Abscisic Acid for Potential Regulation of Tumor Formation in Maize

The infection of maize (Zea mays) by the basidiomycete fungus Ustilago maydis leads to common smut of corn characterized by the production of tumors in susceptible aboveground plant tissues. LC-(ES)MS/MS profiles of abscisic acid (ABA) and 12 different cytokinins (CKs) were determined for infected and uninfected maize tissues over a time course following fungal exposure. Samples were taken at points corresponding to the appearance of disease symptoms. Axenic cultures of haploid and dikaryon forms of U. maydis were also profiled. This study confirmed the capability of Ustilago maydis to synthesize CKs, ABA, and auxin (IAA). It also provided evidence for the involvement of CK and ABA in the U. maydis-maize infection process. Significant quantities of CKs and ABA were detected from axenic cultures of U. maydis as was IAA. CKs and ABA levels were elevated in leaves and stems of maize after infection; notable was the high level of cis-zeatin 9-riboside. Variation among hormone profiles of maize tissues was observed at different time points during infection and between infections with nonpathogenic haploid and pathogenic dikaryon strains. This suggested that CKs and ABA accumulate and are likely metabolized in maize tissue infected with U. maydis. Because U. maydis produced these phytohormones at significant levels, it is possible that the fungal pathogen is a source of these compounds in infected tissue. This is the first study to confirm the production of CKs and document the production of ABA by U. maydis. This study also established an involvement of these phytohormones and a possible functional role for ABA in U. maydis infection of maize.     

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.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.