The secretome of the maize pathogen Ustilago maydis

Ustilago maydis establishes a biotrophic relationship with its host plant, i.e. plant cells stay alive despite massive fungal growth in infected tissue. The genome sequence has revealed that U. maydis is poorly equipped with plant cell wall degrading enzymes and uses novel secreted protein effectors as crucial determinants for biotrophic development. Many of these effector genes are clustered and differentially regulated during plant colonization. In this review, we analyze the secretome of U. maydis by differentiating between secreted enzymes, likely structural proteins of the fungal cell wall (excluding GPI-anchored proteins) as well as likely effectors with either apoplastic or cytoplasmic function. This classification is based on the presence of functional domains, general domain structure and cysteine pattern. In addition, we discuss possible functions of selected protein classes with a special focus on disease development.     

Reprogramming a maize plant: transcriptional and metabolic changes induced by the fungal biotroph Ustilago maydis

The fungal pathogen Ustilago maydis establishes a biotrophic relationship with its host plant maize (Zea mays). Hallmarks of the disease are large plant tumours in which fungal proliferation occurs. Previous studies suggested that classical defence pathways are not activated. Confocal microscopy, global expression profiling and metabolic profiling now shows that U. maydis is recognized early and triggers defence responses. Many of these early response genes are downregulated at later time points, whereas several genes associated with suppression of cell death are induced. The interplay between fungus and host involves changes in hormone signalling, induction of antioxidant and secondary metabolism, as well as the prevention of source leaf establishment. Our data provide novel insights into the complexity of a biotrophic interaction.     

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.     

Carbohydrate involvement during infection of maize by Helminthosporium maydis

Germination and germ tube length of Helminthosporium maydis conidia did not exhibit much difference on fixed decolourized and living green leaves. However, appressoria, penetrations and colonizations were much less on decolourized host leaves and were enhanced significantly when sugars were added in the infection court. Few leached conidia germinated on the decolourized host leaves and appressoria, penetrations and colonizations effected on them by leached conidia were almost negligible. The presence of exogenous sugars and leaf leachates enabled the leached conidia to accomplish some penetrations and colonizations. Carbohydrate content of decolourized leaves and leached conidia was much less than the green leaves and non-leached conidia, respectively. Carbohydrates accumulated at the infection sites/green islands which also exhibited higher chlorophyll content.     

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.     

Ustilago maydis populations tracked maize through domestication and cultivation in the Americas

The domestication of crops and the development of agricultural societies not only brought about major changes in human interactions with the environment but also in plants' interactions with the diseases that challenge them. We evaluated the impact of the domestication of maize from teosinte and the widespread cultivation of maize on the historical demography of Ustilago maydis, a fungal pathogen of maize. To determine the evolutionary response of the pathogen's populations, we obtained multilocus genotypes for 1088 U. maydis diploid individuals from two teosinte subspecies in Mexico and from maize in Mexico and throughout the Americas. Results identified five major U. maydis populations: two in Mexico; two in South America; and one in the United States. The two populations in Mexico diverged from the other populations at times comparable to those for the domestication of maize at 6000-10000 years before present. Maize domestication and agriculture enforced sweeping changes in U. maydis populations such that the standing variation in extant pathogen populations reflects evolution only since the time of the crop's domestication.     

The Ustilago maydis regulatory subunit of a cAMP-dependent protein kinase is required for gall formation in maize.

In the plant, filamentous growth is required for pathogenicity of the corn smut pathogen Ustilago maydis. Earlier, we identified a role for the cAMP signal transduction pathway in the switch between budding and filamentous growth for this fungus. A gene designated ubc1 (for Ustilago bypass of cyclase) was found to be required for filamentous growth and to encode the regulatory subunit of a cAMP-dependent protein kinase (PKA). Here, we show that ubc1 is important for the virulence of the pathogen. Specifically, ubc1 mutants are able to colonize maize plants and, like the wild-type pathogen, cause localized symptoms in association with the presence of hyphae. However, in contrast to plants infected with wild-type cells that often developed galls from initially chlorotic tissue, plants infected with the ubc1 mutant did not produce galls. These data suggest that PKA regulation is critical for the transition from saprophytic to pathogenic growth and from vegetative to reproductive development. Plate mating assays in which exogenous cAMP was applied suggested that the cAMP and b mating-type morphogenetic pathways may be coordinated.     

Infection of maize leaves with Ustilago maydis prevents establishment of C4 photosynthesis

The Basidiomycete fungus Ustilago maydis is the common agent of corn smut and is capable of inducing gall growth on infected tissue of the C4 plant maize (Zea mays). While U. maydis is very well characterized on the genetic level, the physiological changes in the host plant in response to U. maydis infection have not been studied in detail, yet. Therefore, we examined the influence of U. maydis infection on photosynthetic performance and carbon metabolism in maize leaf galls. At all stages of development, U. maydis-induced leaf galls exhibited carbon dioxide response curves, CO2 compensation points and enzymatic activities that are characteristic of C3 photosynthesis, demonstrating that the establishment of C4 metabolism is prevented in infected tissue. Hexose contents and hexose/sucrose ratio of leaf galls remained high at 6 days post infection, while a shift in free sugar metabolism was observed in the uninfected controls at that time point. Concomitantly, transitory starch production and sucrose accumulation during the light period remained low in leaf galls. Given that U. maydis is infectious on young developing tissue, the observed changes in carbohydrate metabolism suggest that the pathogen manipulates the developing leaf tissue to arrest sink-to-source transition in favor of maintaining sink metabolism in the host cells. Furthermore, evidence is presented that carbohydrate supply during the biotrophic phase of the pathogen is assured by a fungal invertase.     

Effects of host plant environment and Ustilago maydis infection on the fungal endophyte community of maize (Zea mays)

The focus of many fungal endophyte studies has been how plants benefit from endophyte infection. Few studies have investigated the role of the host plant as an environment in shaping endophyte community diversity and composition. The effects that different attributes of the host plant, that is, host genetic variation, host variation in resistance to the fungal pathogen Ustilago maydis and U. maydis infection, have on the fungal endophyte communities in maize (Zea mays) was examined. The internal transcribed spacer (ITS) region of the rDNA was sequenced to identify fungi and the endophyte communities were compared in six maize lines that varied in their resistance to U. maydis. It was found that host genetic variation, as determined by maize line, had significant effects on species richness, while the interactions between line and U. maydis infection and line and field plot had significant effects on endophyte community composition. However, the effects of maize line were not dependent on whether lines were resistant or susceptible to U. maydis. Almost 3000 clones obtained from 58 plants were sequenced to characterize the maize endophyte community. These results suggest that the endophyte community is shaped by complex interactions and factors, such as inoculum pool and microclimate, may be important.     

Ortholog of BRCA2-interacting protein BCCIP controls morphogenetic responses during DNA replication stress in Ustilago maydis

The BRCA2 tumor suppressor functions in repair of DNA by homologous recombination through regulating the action of Rad51. In turn, BRCA2 appears to be regulated by other interacting proteins. Dss1, a small interacting protein that binds to the C-terminal domain, has a profound effect on activity as deduced from studies on the BRCA2-related protein Brh2 in Ustilago maydis. Evidence accumulating in mammalian systems suggests that BCCIP, another small interacting protein that binds to the C-terminal domain of BRCA2, also serves to regulate homologous recombination activity. Here we were interested in testing the role of the putative U. maydis BCCIP ortholog Bcp1 in DNA repair and recombination. In keeping with the mammalian paradigm, Bcp1 bound to the C-terminal region of Brh2. Mutants deleted of the gene were extremely slow growing, showed a delay passing through S phase and exhibited sensitivity to hydroxyurea, but were otherwise normal in DNA repair and homologous recombination. In the absence of Bcp1 cells were unable to maintain the wild type morphology when challenged by a DNA replication stress. These results suggest that Bcp1 could be involved in coordinating morphogenetic events with DNA processing during replication.