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

Systematic Y2H screening reveals extensive effector-complex formation

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

Efficient screening for virulence factors with mutant pools in the Ustilago maydis – Zea mays pathosystem

Biotrophe und filamentöse Pathogene stellen eine substanzielle Gefahr für pflanzliche Erträge dar und verursachen der Landwirtschaft jährlich beträchtliche Verluste. Die Virulenz der Pathogene beruht auf kleinen, sekretierten Molekülen, weitestgehend bekannt als Effektoren. In genomischen Analysen konnte gezeigt werden, dass filamentöse Pathogene große Arsenale an Effektoren haben, von denen die meisten keine bekannten Proteindomänen besitzen, die Aufschluss auf die Funktion der Effektoren geben könnten. Deletionsmutanten von Effektoren, die einen starken Einfluss auf die Virulenz des Pathogens haben, weisen meist auch einen verminderten Virulenzphänotyp auf. Im Rahmen dieser Dissertation wurde die Technik namens „insertion Pool-Sequencing“ (iPool-Seq) entwickelt, die es ermöglicht, effizient und im Hochdurchsatz Effektoren zu identifizieren, die einen Beitrag zur Virulenz leisten. Die Technik ermöglicht die Analyse von Infektionen mit mehreren Pathogenmutanten gleichzeitig und basiert letztendlich auf Hochdurchsatzsequenzierungen von Mutanten Genomen. iPool-Seq ist besonders effizient, wodurch erfolgreich die in der genomischen DNS enthaltenen Sequenzflanken der Insertionskassetten direkt aus dem infizierten Pflanzenmaterial angereichert werden können. In dieser Arbeit wurde iPool-Seq an einer Mutantensammlung des Maispathogens Ustilago maydis getestet und reproduzierbare sowie quantitative auswertbare Sequenzen erhalten. Unter den 28 identifizierten Mutanten, die eine signifikant verminderte Virulenz aufzeigten, konnten 5 bekannte Mutanten verifiziert werden. Das Verfahren istmit jeder Sammlung von Insertionsmutanten kompatibel und könnte zum Beispiel auch für die Entschlüsselung von essentiellen Genen von Mikroben verwendet werden. Des Weiteren wurde im Rahmen dieser Doktorarbeit eine Kategorisierung von möglichen Funktionsweisen von Effektoren vorgeschlagen: Effektoren können auf das Pathogen selbst Auswirkungen haben, oder eine unterdrückende oder aktivierende Funktion innerhalb der Pflanze einnehmen. Die systematische und eindeutige Entschlüsselung von Funktionsweisen der Effektoren in filamentösen Pathogenen ist eines der Hauptherausforderungen im Feld der Pflanzen-Mikroben Interaktionen. Diese funktionellen Analysen geben Aufschluss über potentielle pflanzliche Interaktionspartner, die subzelluläre Lokalisation in der Pflanze und den Beitrag der Virulenz eines Effektors. Bei erfolgreicher und umfassender Analyse der Funktionen von Effektoren kann dieses Wissen zu einer Weiterentwicklung von resistenten Nutzpflanzen eingesetzt werden.Biotrophic, filamentous plant pathogens are a substantial threat to plant yield and cause immense annual losses in agriculture. Their virulence is promoted by small, secreted molecules, commonly known as effectors. Genomic analysis revealed that filamentous pathogens have large arsenals of effectors, mostly lacking known domains that could indicate their function. Effectors that have a strong impact on virulence likely display a reduced virulence phenotype upon genomic deletion. To test this efficiently and in high-throughput, developed insertion Pool-Sequencing (iPool-Seq) was developed, a tool that allows for the analysis of insertional mutant pool infections by extensive parallel Illumina sequencing. iPool-Seq is extremely sensitive, enabling genomic DNA extractions coupled with efficient enrichment of genome-insertion site junctions directly from in vivo infected host material. iPool-Seq was tested on an insertional mutant library of the maize-pathogen Ustilago maydis, yielding highly reproducible and quantitative results. Among the identified virulence factors, iPool-Seq confirmed five well characterized mutants and identified 23 unknown virulence factors. The iPool-Seq protocol is compatible with any existing insertional mutant library and is a promising tool that is not restricted to effector biology but has the potential to elucidate essential genes of various microbes. Moreover, a functional categorization was proposed, wherein effectors can act as self-modifiers, or either as suppressors, or activators of plant host targets. In future, it is of outstanding interest to decipher effector functions on a genome wide level with high precision. These functional analyses comprise effector host target identification, in planta subcellular localization and contribution of effectors to virulence. This knowledge might foster engineering of more resistant crop varieties in future

Strategies for successful host invasion.

<p>Plant-colonizing microbes employ effectors fulfilling various functions during the host invasion, which are visualized symbolically in this cartoon. Different modes of action (self-binding and self-modifying, activating or inhibiting activities) of effectors described in the text may be applied to serve the listed strategies (text on grey oval background).</p

Tetracycline-controlled (TetON) gene expression system for the smut fungus Ustilago maydis

Ustilago maydis is a biotrophic phytopathogenic fungus that causes corn smut disease. As a well-established model system, U. maydis is genetically fully accessible with large omics datasets available and subject to various biological questions ranging from DNA-repair, RNA-transport, and protein secretion to disease biology. For many genetic approaches, tight control of transgene regulation is important. Here we established an optimised version of the Tetracycline-ON (TetON) system for U. maydis. We demonstrate the Tetracycline concentration-dependent expression of fluorescent protein transgenes and the system’s suitability for the induced expression of the toxic protein BCL2 Associated X-1 (Bax1). The Golden Gate compatible vector system contains a native minimal promoter from the mating factor a-1 encoding gene, mfa with ten copies of the tet-regulated operator (tetO) and a codon optimised Tet-repressor (tetR*) which is translationally fused to the native transcriptional corepressor Mql1 (UMAG_05501). The metabolism-independent transcriptional regulator system is functional both, in liquid culture as well as on solid media in the presence of the inducer and can become a useful tool for toxin-antitoxin studies, identification of antifungal proteins, and to study functions of toxic gene products in Ustilago maydis

In vivo insertion pool sequencing identifies virulence factors in a complex fungal–host interaction

<div><p>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 <i>U</i>. <i>maydis</i>. 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.</p></div

iPool-Seq library preparation workflow features tagmentation and UMIs.

<p>(<b>a</b>) Library preparation was carried out for the input mutant collection and for the output after infection. For the output, we harvested infected areas of the second and third maize leaves and isolated gDNA. (<b>b</b>) Extracted gDNA was fragmented with Tn5 Transposase loaded with custom adapters containing an SBS (green), 12-bp UMI, and Tn5 hyperactive MEs (blue). Genome–hpt resistance cassette junctions were PCR-amplified with biotinylated primers directed against UPSs (magenta) and adapter-specific primers directed at the SBS. (<b>c</b>) Biotinylated PCR products were streptavidin-affinity–purified and Illumina-compatible P5 (purple; NGS1) and P7 (purple; NGS2) ends were introduced by nested PCR. Final products were subjected to Illumina PE sequencing on a MiSeq platform. gDNA, genomic DNA; hpt, hygromycin phosphotransferase; iPool-Seq, insertion Pool-Sequencing ME, mosaic end; PE, paired-end; ROI, region of interest; SBS, sequencing primer binding site; UMI, unique molecular identifier; UPS, unique primer binding site.</p

Many ways to TOPLESS - manipulation of plant auxin signalling by a cluster of fungal effectors

Plant biotrophic pathogens employ secreted molecules, called effectors, to suppress the host immune system and redirect the host's metabolism and development in their favour. Putative effectors of the gall-inducing maize pathogenic fungus Ustilago maydis were analysed for their ability to induce auxin signalling in plants. Using genetic, biochemical, cell-biological, and bioinformatic approaches we functionally elucidate a set of five, genetically linked effectors, called Topless (TPL) interacting protein (Tips) effectors that induce auxin signalling. We show that Tips induce auxin signalling by interfering with central corepressors of the TPL family. CRISPR-Cas9 mutants and deletion strain analysis indicate that the auxin signalling inducing subcluster effectors plays a redundant role in virulence. Although none of the Tips seem to have a conserved interaction motif, four of them bind solely to the N-terminal TPL domain and, for Tip1 and Tip4, we demonstrate direct competition with auxin/indole-3-acetic acid transcriptional repressors for their binding to TPL class of corepressors. Our findings reveal that TPL proteins, key regulators of growth-defence antagonism, are a major target of the U. maydis effectome

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

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