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.DOI: http://dx.doi.org/10.7554/eLife.20522.001

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 Ustilago maydis (U. maydis) is the causal agent of maize smut disease. During the colonization process, the fungus secretes effector proteins which suppress immune responses and redirect the host metabolism in favor of the pathogen. As effectors play a critical role during plant colonization, their identification and functional characterization is essential to understanding biotrophy and disease.  Using biochemical, molecular, and transcriptomic techniques, we performed a functional characterization of the U. maydis effector Jasmonate/Ethylene signaling inducer 1 (Jsi1).  Jsi1 interacts with several members of the plant co-repressor family Topless/Topless related (TPL/TPR). Jsi1 expression in Zea mays (Z. mays) and Arabidopsis thaliana (A. thaliana) leads to transcriptional induction of the ethylene response factor (ERF) branch of the jasmonate/ethylene (JA/ET) signaling pathway. In A. thaliana, activation of the ERFbranch leads to biotrophic susceptibility. Jsi1 likely activates the ERF-branch via an EAR motif, which resembles EAR motifs from plant ERF transcription factors, that interacts with TPL/TPR proteins.  EAR motif-containing effector candidates were identified from different fungal species including Magnaporthe oryzae, Sporisorium scitamineum, and Sporisorium reilianum. Interaction between plant TPL proteins and these effector candidates from biotrophic and hemibiotrophic fungi indicates the convergent evolution of effectors modulating the TPL/TPR co-repressor hub.

Translation termination requires eRF1 and eRF3 for polypeptide- and tRNA-release on stop codons. Additionally, Dbp5/DDX19 and Rli1/ABCE1 are required; however, their function in this process is currently unknown. Using a combination of in vivo and in vitro experiments, we show that they regulate a stepwise assembly of the termination complex. Rli1 and eRF3-GDP associate with the ribosome first. Subsequently, Dbp5-ATP delivers eRF1 to the stop codon and in this way prevents a premature access of eRF3. Dbp5 dissociates upon placing eRF1 through ATP-hydrolysis. This in turn enables eRF1 to contact eRF3, as the binding of Dbp5 and eRF3 to eRF1 is mutually exclusive. Defects in the Dbp5-guided eRF1 delivery lead to premature contact and premature dissociation of eRF1 and eRF3 from the ribosome and to subsequent stop codon readthrough. Thus, the stepwise Dbp5-controlled termination complex assembly is essential for regular translation termination events. Our data furthermore suggest a possible role of Dbp5/DDX19 in alternative translation termination events, such as during stress response or in developmental processes, which classifies the helicase as a potential drug target for nonsense suppression therapy to treat cancer and neurodegenerative diseases.

The smut fungus Sporisorium reilianum occurs in two varieties (S. reilianum f. sp. reilianum and S. reilianum f. sp. zeae) that cause head smut disease on sorghum and maize, respectively. Prior to plant infection, compatible haploid sporidia of S. reilianum fuse to form infectious dikaryotic hyphae that penetrate the leaf surface, spread throughout the plant, and reach the inflorescences, in which spore formation occurs. To elucidate the basis of host specificity of the two S. reilianum varieties, we compared disease etiology of S. reilianum f. sp. reilianum and S. reilianum f. sp. zeae on sorghum and maize. Both varieties could penetrate and multiply in both hosts. However, red spots appeared on inoculated leaves after sorghum infection with S. reilianum f. sp. zeae. Using matrix-assisted laser desorption-ionization time of flight analysis of leaf extracts, we show that sorghum reacts with the production of the red and orange phytoalexins luteolinidin and apigeninidin upon colonization by S. reilianum f. sp. zeae but not by S. reilianum f. sp. reilianum. Using in vitro growth assays, we demonstrate that luteolinidin but not apigeninidin slows vegetative growth of both S. reilianum f. sp. zeae and S. reilianum f. sp. reilianum. However, the phytoalexin biosynthesis gene SbDFR3 is only induced in sorghum after infection with S. reilianum f. sp. zeae, as shown by quantitative real-time polymerase chain reaction. This suggests that regulation of luteolinidin biosynthesis determines infection success of S. reilianum on sorghum.

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.

Large-scale insertional mutagenesis screens can be powerful genome-wide tools if they are streamlined with efficient downstream analysis, which is a serious bottleneck in complex biological systems. A major impediment to the success of next-generation sequencing (NGS)-based screens for virulence factors is that the genetic material of pathogens is often underrepresented within the eukaryotic host, making detection extremely challenging. We therefore established insertion Pool-Sequencing (iPool-Seq) on maize infected with the biotrophic fungus U. maydis. iPool-Seq features tagmentation, unique molecular barcodes, and affinity purification of pathogen insertion mutant DNA from in vivo-infected tissues. In a proof of concept using iPool-Seq, we identified 28 virulence factors, including 23 that were previously uncharacterized, from an initial pool of 195 candidate effector mutants. Because of its sensitivity and quantitative nature, iPool-Seq can be applied to any insertional mutagenesis library and is especially suitable for genetically complex setups like pooled infections of eukaryotic hosts.

During infection pathogens secrete small molecules, termed effectors, to manipulate and control the interaction with their specific hosts. Both the pathogen and the plant are under high selective pressure to rapidly adapt and co-evolve in what is usually referred to as molecular arms race. Components of the host’s immune system form a network that processes information about molecules with a foreign origin and damage-associated signals, integrating them with developmental and abiotic cues to adapt the plant’s responses. Both in the case of nucleotide-binding leucine-rich repeat receptors and leucine-rich repeat receptor kinases interaction networks have been extensively characterized. However, little is known on whether pathogenic effectors form complexes to overcome plant immunity and promote disease. Ustilago maydis, a biotrophic fungal pathogen that infects maize plants, produces effectors that target hubs in the immune network of the host cell. Here we assess the capability of U. maydis effector candidates to interact with each other, which may play a crucial role during the infection process. Using a systematic yeast-two-hybrid approach and based on a preliminary pooled screen, we selected 63 putative effectors for one-on-one matings with a library of nearly 300 effector candidates. We found that 126 of these effector candidates interacted either with themselves or other predicted effectors. Although the functional relevance of the observed interactions remains elusive, we propose that the observed abundance in complex formation between effectors adds an additional level of complexity to effector research and should be taken into consideration when studying effector evolution and function. Based on this fundamental finding, we suggest various scenarios which could evolutionarily drive the formation and stabilization of an effector interactome.