Overrepresentation Analyses of Differentially Expressed Genes in the Smut Fungus Ustilago bromivora during Saprophytic and in planta Growth.

We have established the Ustilago bromivora-Brachypodium spp. interaction as a new model pathosystem for biotrophic fungal plant infections of the head smut type (Rabe et al., 2016). In this protocol, the methodology used for comparing gene expression between saprophytic and in planta growth of the fungus is described. The experimental and analytical pipeline, how next generation RNA sequencing (Illumina RNA-Seq) analysis can be used to obtain lists of genes significantly up or down regulated in planta in comparison to axenic culture is given. Furthermore, different methods to identify functional categories that are over- or under-represented among specific classes of genes are presented.

A complete toolset for the study of Ustilago bromivora and Brachypodium sp. as a fungal-temperate grass pathosystem.

Due to their economic relevance, the study of plant pathogen interactions is of importance. However, elucidating these interactions and their underlying molecular mechanisms remains challenging since both host and pathogen need to be fully genetically accessible organisms. Here we present milestones in the establishment of a new biotrophic model pathosystem: Ustilago bromivora and Brachypodium sp. We provide a complete toolset, including an annotated fungal genome and methods for genetic manipulation of the fungus and its host plant. This toolset will enable researchers to easily study biotrophic interactions at the molecular level on both the pathogen and the host side. Moreover, our research on the fungal life cycle revealed a mating type bias phenomenon. U. bromivora harbors a haplo-lethal allele that is linked to one mating type region. As a result, the identified mating type bias strongly promotes inbreeding, which we consider to be a potential speciation driver.

Phytohormone sensing in the biotrophic fungus Ustilago maydis - the dual role of the transcription factor Rss1.

The phenolic compound salicylic acid (SA) is a key signalling molecule regulating local and systemic plant defense responses, mainly against biotrophs. Many microbial organisms, including pathogens, share the ability to degrade SA. However, the mechanism by which they perceive SA is unknown. Here we show that Ustilago maydis, the causal agent of corn smut disease, employs a so far uncharacterized SA sensing mechanism. We identified and characterized the novel SA sensing regulator, Rss1, a binuclear zinc cluster protein with dual functions as putative SA receptor and transcriptional activator regulating genes important for SA and tryptophan degradation. Rss1 represents a major component in the identified SA sensing pathway during the fungus' saprophytic stage. However, Rss1 does not have a detectable impact on virulence. The data presented in this work indicate that alternative or redundant sensing cascades exist that regulate the expression of SA-responsive genes in U. maydis during its pathogenic development.

Degradation of the plant defence hormone salicylic acid by the biotrophic fungus Ustilago maydis.

Salicylic acid (SA) is a key plant defence hormone which plays an important role in local and systemic defence responses against biotrophic pathogens like the smut fungus Ustilago maydis. Here we identified Shy1, a cytoplasmic U. maydis salicylate hydroxylase which has orthologues in the closely related smuts Ustilago hordei and Sporisorium reilianum. shy1 is transcriptionally induced during the biotrophic stages of development but not required for virulence during seedling infection. Shy1 activity is needed for growth on plates with SA as a sole carbon source. The trigger for shy1 transcriptional induction is SA, suggesting the possibility of a SA sensing mechanism in this fungus.

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.