Interspecific Sex in Grass Smuts and the Genetic Diversity of Their Pheromone-Receptor System

The grass smuts comprise a speciose group of biotrophic plant parasites, so-called Ustilaginaceae, which are specifically adapted to hosts of sweet grasses, the Poaceae family. Mating takes a central role in their life cycle, as it initiates parasitism by a morphological and physiological transition from saprobic yeast cells to pathogenic filaments. As in other fungi, sexual identity is determined by specific genomic regions encoding allelic variants of a pheromone-receptor (PR) system and heterodimerising transcription factors. Both operate in a biphasic mating process that starts with PR–triggered recognition, directed growth of conjugation hyphae, and plasmogamy of compatible mating partners. So far, studies on the PR system of grass smuts revealed diverse interspecific compatibility and mating type determination. However, many questions concerning the specificity and evolutionary origin of the PR system remain unanswered. Combining comparative genetics and biological approaches, we report on the specificity of the PR system and its genetic diversity in 10 species spanning about 100 million years of mating type evolution. We show that three highly syntenic PR alleles are prevalent among members of the Ustilaginaceae, favouring a triallelic determination as the plesiomorphic characteristic of this group. Furthermore, the analysis of PR loci revealed increased genetic diversity of single PR locus genes compared to genes of flanking regions. Performing interspecies sex tests, we detected a high potential for hybridisation that is directly linked to pheromone signalling as known from intraspecies sex. Although the PR system seems to be optimised for intraspecific compatibility, the observed functional plasticity of the PR system increases the potential for interspecific sex, which might allow the hybrid-based genesis of newly combined host specificities

mRNA Inventory of Extracellular Vesicles from Ustilago maydis

Extracellular vesicles (EVs) can transfer diverse RNA cargo for intercellular communication. EV-associated RNAs have been found in diverse fungi and were proposed to be relevant for pathogenesis in animal hosts. In plant-pathogen interactions, small RNAs are exchanged in a cross-kingdom RNAi warfare and EVs were considered to be a delivery mechanism. To extend the search for EV-associated molecules involved in plant-pathogen communication, we have characterised the repertoire of EV-associated mRNAs secreted by the maize smut pathogen, Ustilago maydis. For this initial survey, we examined EV-enriched fractions from axenic filamentous cultures that mimic infectious hyphae. EV-associated RNAs were resistant to degradation by RNases and the presence of intact mRNAs was evident. The set of mRNAs enriched inside EVs relative to the fungal cells are functionally distinct from those that are depleted from EVs. mRNAs encoding metabolic enzymes are particularly enriched. Intriguingly, mRNAs of some known effectors and other proteins linked to virulence were also found in EVs. Furthermore, several mRNAs enriched in EVs are also upregulated during infection, suggesting that EV-associated mRNAs may participate in plant-pathogen interactions

Cytoplasmic Transport Machinery of the SPF27 Homologue Num1 in Ustilago maydis

In the phytopathogenic basidiomycete Ustilago maydis, the Num1 protein has a pivotal function in hyphal morphogenesis. Num1 functions as a core component of the spliceosome-associated Prp19/CDC5 complex (NTC). The interaction of Num1 with the kinesin motor Kin1 suggests a connection between a component of the splicing machinery and cytoplasmic trafficking processes. Previously it was shown that Num1 localizes predominantly in the nucleus; however, due to the diffraction-limited spatial resolution of conventional optical microscopy, it was not possible to attribute the localization to specific structures within the cytoplasm. We have now employed super-resolution localization microscopy to visualize Num1 in the cytoplasm by fusing it to a tandem dimeric Eos fluorescent protein (tdEosFP). The Num1 protein is localized within the cytoplasm with an enhanced density in the vicinity of microtubules. Num1 movement is found predominantly close to the nucleus. Movement is dependent on its interaction partner Kin1, but independent of Kin3. Our results provide strong evidence that, in addition to its involvement in splicing in the nucleus, Num1 has an additional functional role in the cytosol connected to the Kin1 motor protein

Health-Related Quality of Life and Mental Health after Surgical Treatment of Hepatocellular Carcinoma in the Era of Minimal-Invasive Surgery: Resection versus Transplantation

Laparoscopic liver resection (LLR) is an increasingly relevant treatment option for patients with resectable hepatocellular carcinoma (HCC). Orthotopic liver transplantation (OLT) has been considered optimal treatment for HCC in cirrhosis, but is challenged by rising organ scarcity. While health-related quality of life (HRQoL) and mental health are well-documented after OLT, little is known about HRQoL in HCC patients after LLR. We identified all HCC patients who underwent LLR at our hospital between 2014 and 2018. HRQoL and mental health were assessed using the Short Form 36 and the Hospital Anxiety and Depression Scale, respectively. Outcomes were compared to a historic cohort of HCC patients after OLT. Ninety-eight patients received LLR for HCC. Postoperative morbidity was 25% with 17% minor complications. LLR patients showed similar overall HRQoL and mental health to OLT recipients, except for lower General Health (p = 0.029) and higher anxiety scores (p = 0.010). We conclude that LLR can be safely performed in patients with HCC, with or without liver cirrhosis. The postoperative HRQoL and mental health are comparable to that of OLT recipients in most aspects. LLR should thus always be considered an alternative to OLT, especially in times of organ shortage

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens. ©2006 Nature Publishing Group.J.K., M. B. and R.K. thank G. Sawers and U. Kämper for critical reading of the manuscript. The genome sequencing of Ustilago maydis strain 521 is part of the fungal genome initiative and was funded by National Human Genome Research Institute (USA) and BayerCropScience AG (Germany). F.B. was supported by a grant from the National Institutes of Health (USA). J.K. and R.K. thank the German Ministry of Education and Science (BMBF) for financing the DNA array setup and the Max Planck Society for their support of the manual genome annotation. F.B. was supported by a grant from the National Institutes of Health, B.J.S. was supported by the Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation, J.W.K. received funding from the Natural Sciences and Engineering Research Council of Canada, J.R.-H. received funding from CONACYT, México, A.M.-M. was supported by a fellowship from the Humboldt Foundation, and L.M. was supported by an EU grant. Author Contributions All authors were involved in planning and executing the genome sequencing project. B.W.B., J.G., L.-J.M., E.W.M., D.D., C.M.W., J.B., S.Y., D.B.J., S.C., C.N., E.K., G.F., P.H.S., I.H.-H., M. Vaupel, H.V., T.S., J.M., D.P., C.S., A.G., F.C. and V. Vysotskaia contributed to the three independent sequencing projects; M.M., G.M., U.G., D.H., M.O. and H.-W.M. were responsible for gene model refinement, database design and database maintenance; G.M., J. Kämper, R.K., G.S., M. Feldbrügge, J.S., C.W.B., U.F., M.B., B.S., B.J.S., M.J.C., E.C.H.H., S.M., F.B., J.W.K., K.J.B., J. Klose, S.E.G., S.J.K., M.H.P., H.A.B.W., R.deV., H.J.D., J.R.-H., C.G.R.-P., L.O.-C., M.McC., K.S., J.P.-M., J.I.I., W.H., P.G., P.S.-A., M. Farman, J.E.S., R.S., J.M.G.-P., J.C.K., W.L. and D.H. were involved in functional annotation and interpretation; T.B., O.M., L.M., A.M.-M., D.G., K.M., N.R., V. Vincon, M. VraneŠ, M.S. and O.L. performed experiments. J. Kämper, R.K. and M.B. wrote and edited the paper with input from L.-J.M., J.G., F.B., J.W.K., B.J.S. and S.E.G. Individual contributions of authors can be found as Supplementary Notes

Das AUACCC-bindende Protein Khd4 kontrolliert Morphogenese und Pathogenität in Ustilago maydis

Der phytopathogene Basidiomyzet Ustilago maydis ist der Erreger des Maisbeulenbrandes. Für eine erfolgreiche Infektion der Wirtspflanze Zea mays ist die Bildung eines dikaryotischen Filaments erforderlich. Erst in diesem Stadium kann der Pilz die Pflanzenoberfläche penetrieren und die Tumorbildung induzieren. U. maydis stellt nicht nur ein Modellsystem für die Pilz/Wirt-Interaktion dar, sondern wurde in der Vergangenheit bspw. auch für Untersuchungen der DNA-Reparatur oder der RNA-Biologie genutzt. Erste Charakterisierungen des Einflusses RNA-bindender Proteine auf die Pathogenität deuteten dabei an, dass die posttranskriptionelle Regulation innerhalb des infektiösen Stadiums eine bedeutende Rolle spielt. Jedoch wurden bisher nur wenige RNA-bindende Proteine hinsichtlich ihrer regulatorischen Funktion in filamentösen Pilzen näher charakterisiert. In dieser Arbeit wurde die Rolle des RNA-bindenden Proteins Khd4 von U. maydis detailliert untersucht. Die Deletion des Gens führt zu einem pleiotropen Phänotyp, der sich in einem Zytokinesedefekt haploider Sporidien, reduzierter Filamentbildung und stark verringerter Virulenz äußert. Um die Funktion von Khd4 hinsichtlich dessen Einfluss auf Morphologie und Pathogenität zu verstehen, wurde die RNA/Protein-Interaktion charakterisiert und mögliche Ziel-Transkripte identifiziert. In dieser Dissertation konnte gezeigt werden, dass die Tandem-KH-Domänen 3 und 4 essentiell für die Funktion von Khd4 und für die RNA-Bindung in vivo sind. Mutationen wichtiger Aminosäuren innerhalb dieser Domänen äußerten sich in einem dem khd4Δ-ähnlichen Phänotyp und verhinderten die Bindung AUACCC-enthaltender RNA, welches mit Hilfe des Hefe-Drei-Hybrid-Systems ermittelt werden konnte. Die Anwesenheit des cis-aktiven Elements AUACCC war dabei sowohl notwendig als auch hinreichend. Um zu untersuchen, ob es sich bei diesem Motiv um ein regulatorisches RNA-Element in U. maydis handelt, wurden zwei unabhängige Analysen durchgeführt. Zum einen wurde über Microarray-Analysen ermittelt, dass das Motiv AUACCC in differentiell regulierten mRNAs angereichert war (29 von 72 regulierten mRNAs). Über bioinformatische Analysen wurde zum anderen eine signifikante Anreicherung des Motivs in den ersten 150 Nukleotiden der 3untranslatierten Region (UTR) festgestellt. Die Untersuchung experimentell ermittelter 3UTR-Längen bestätigte dabei diese Anreicherung. Weiterhin war die überwiegende Mehrheit der differentiell regulierten Transkripte im khd4-Deletionsstamm hochreguliert, was für eine destabilisierende Funktion von Khd4 sprach. Unterstützt wurde diese Hypothese durch die partielle Kolokalisation von Khd4 mit processing bodies (prozessierenden Körpern, P-bodies), welche in mRNA-Abbauprozessen von hoher Bedeutung sind. Die 29 AUACCC-enthaltenden, differentiell deregulierten Transkripte stellen außerdem direkte Ziel-Transkripte dar, welche einen Einfluss auf den Phänotyp des khd4Δ-Stammes besitzen könnten. Folglich scheint die RNA-Bindung von Khd4 essentiell zu sein, um posttranskriptionell AUACCC-enthaltende mRNAs durch Rekrutierung von P-bodies zu degradieren. Mit Hilfe der Khd4-abhängigen Regulation wird dadurch möglicherweise die korrekte Entwicklung des haploiden und infektiösen Stadiums von U. maydis gewährleistet

Biotrophic Development of Ustilago maydis and the Response of Its Host Plant Maize

Fungal plant pathogens affect the quality of food and feed produced from infected plants and cause substantial yield losses every year. Especially fungi infecting cereal crops represent an ernormous thread. The biotrophic fungus Ustilago maydis is the causative agent of the smut disease on maize. Molecular pathways essential for the initiation of fungal pathogenicity, like mating of two compatible sporidia, the establishment of an infectious dikaryon and the penetration process leading to plant infection are intensively studied in U. maydis. However, the strategies used by the fungus to proliferate within the plant and to deal with the hostile environment, are vastly unknown. This dissertation investigates the complex molecular interplay between Ustilago maydis and its host plant in more detail, focusing on three different aspects. In U. maydis the initiation of sexual development and pathogenicity is controlled by two homedomain proteins bE and bW, which form an active transcription factor after fusion of two compatible sporidia. By constructing temperature-sensitive bE proteins, I was able to demonstrate that also the proliferation of U. maydis within the plant is regulated by the b mating type transcription factor (2.1). The inactivation of the bW/bE complex within the plant stops fungal development and leads to the deregulation of secreted proteins, which are believed to interfere with plant defense responses. U. maydis establishes a compatible biotrophic relationship with its host. To analyze the plant cell responses towards this forced interaction, global expression analysis and metabolic profiling were performed monitoring a time-course of infection (2.2). Expression analyses revealed an initial recognition of U. maydis by the maize plant, leading to the induction of basal plant defense responses. After U. maydis has penetrated the plant these defense responses are suppressed, suggesting an active interference with the plant immune system. Moreover, during disease progression U. maydis infected maize leaves do not develop into photosynthetically active source tissues, but maintain the characteristics of a nutrient sink. Like typical plant nutrient sinks the infected area is supplied with sucrose that is feeding the fungus. As nutrient availability determines the fitness of the pathogen, it also determines the pathogens success to conquer the plant. Thus, biotrophic fungi like U. maydis have to develop strategies to feed on nutrients provided by a living host plant. By identifying two U. maydis sugar transporters, Srt1 and Hxt1, as necessary for full fungal virulence, I was able to analyze which plant-derived carbohydrates are crucial for biotrophic development (2.3; 2.4). Srt1, a novel kind of sucrose transporter, is exclusively expressed during infection. Its unusual high sucrose affinity is well suited to compete with plant-derived sucrose uptake systems at the plant/fungus interfacen (2.3). Hxt1 utilizes hexoses glucose, fructose and mannose, and with lower affinity also galactose and xylose. Deletion of hxt1 reduces fungal pathogenicity, influences growth and hampers monosaccharide-dependent gene regulation. Moreover, expression analysis revealed that Hxt1 has a dual function as monosaccharide-transporter and -sensor (2.4). As double-deletion mutants of hxt1 and srt1 fail to induce severe disease symptoms, both uptake of sucrose and its cleavage products glucose and fructose are crucial for in planta development of U. maydis (2.4). U. maydis is recognized by the maize plant already prior to infection, resulting in the induction of basal plant defense responses. However, as soon as the fungus penetrates the plant these defense responses are manipulated by U. maydis, most probably caused by the action of fungal secreted proteins interfering with recognition and defense pathways. During disease progression, the infected maize tissue remains a sucrose-dependent nutrient sink, which lacks photosynthetic activity. This sink supplies U. maydis with sucrose and hexoses utilized by Srt1 and Hxt1 to promote fungal growth. Initiation and maintenance of the biotrophic interaction, including the expression of secreted proteins necessary to manipulate the host, are regulated by a complex transcription cascade, which is controlled by the bE/bW heterodimer. The b-cascade not only regulates fungal proliferation and differentiation, but also adapts the fungal needs towards changing plant tissues

Die Identifikation von Ziel-Transkripten des RNA bindenden Proteins Rrm4 aus Ustilago maydis

Die Etablierung einer zellulÄren Polaritätsachse ist Voraussetzung für filamentöses Wachstum in Pilzen. Aufbau und Aufrechterhaltung zugrunde liegender molekularer Gradienten werden häufig durch aktiven subzellulären Transport entlang des Zytoskeletts gewährleistet, wobei als Fracht Proteine, Vesikel sowie mRNA befördert werden. In Ustilago maydis ist das putativ RNA bindende Protein Rrm4 Teil von Partikeln, die sich bidirektional entlang von Mikrotubuli im Filament bewegen. Der Transportprozess sowie die im Protein enthaltenen RNA-Erkennungs-Motive (RRM) sind wichtig für die akkurate Bildung der Polaritätsachse und die Pathogenität des Pilzes. Ziel dieser Arbeit war es, Transkripte zu identifizieren, die von Rrm4 im Filament gebunden werden. Mit Hilfe der CLIP-Technologie (ultraviolet crosslinking and immunoprecipitation) konnte zunächst gezeigt werden, dass Rrm4 in vivo mit RNA interagierte. Diese Bindung erfolgte über die RRM-Domänen des Proteins. Nach Aufreinigung wurden 55 durch Rrm4 gebundene Transkripte identifiziert. Eine bioinformatische Analyse der erhaltenen Sequenzen ergab CA reiche Elemente als mögliches sequenzspezifisches Bindemotiv. Interessanterweise kodierte ein Teil der Ziel-Transkripte für Proteine, deren Homologe in anderen Organismen an polaren Wachstumsprozessen beteiligt sind. Für die Analyse der subzellulären Lokalisation der Transkripte wurde die FISH-Technologie (fluorescence in situ hybridisation) in U.maydis-Filamenten etabliert. Die Quantifizierung der Signalverteilung erfolgte mit dem dafür implementierten Programm PIA (peak-identifying algorithm). Am Beispiel der mRNA des Septins Cdc3 konnte demonstriert werden, dass diese in Partikeln akkumulierte, deren Bildung von Rrm4 abhängig war. Die durch den Transport dieser Partikel erreichte Verteilung der mRNA könnte wichtig sein, um auch vom Kern entferntere Bereiche des Filaments zu versorgen. Entsprechend korrelierte der nahezu vollständige Verlust der mRNA-Partikel in rrm4-Deletionsstämmen mit einer stark reduzierten Akkumulation des Cdc3-Proteins in den Filamentspitzen. Diese Ergebnisse deuteten an, dass der Rrm4 abhängige Transport der cdc3-mRNA entscheidend für die korrekte Verteilung des entsprechenden Proteins in der Hyphe sein könnte. Durch die Lokalisierung dieses und möglicher weiterer Polaritätsfaktoren könnte die Rrm4 vermittelte posttranskriptionelle Regulation einen steuernden Einfluss auf polare Wachstumsprozesse nehmen

The ESCRT regulator Did2 maintains the balance between long-distance endosomal transport and endocytic trafficking.

In highly polarised cells, like fungal hyphae, early endosomes function in both endocytosis as well as long-distance transport of various cargo including mRNA and protein complexes. However, knowledge on the crosstalk between these seemingly different trafficking processes is scarce. Here, we demonstrate that the ESCRT regulator Did2 coordinates endosomal transport in fungal hyphae of Ustilago maydis. Loss of Did2 results in defective vacuolar targeting, less processive long-distance transport and abnormal shuttling of early endosomes. Importantly, the late endosomal protein Rab7 and vacuolar protease Prc1 exhibit increased shuttling on these aberrant endosomes suggesting defects in endosomal maturation and identity. Consistently, molecular motors fail to attach efficiently explaining the disturbed processive movement. Furthermore, the endosomal mRNP linker protein Upa1 is hardly present on endosomes resulting in defects in long-distance mRNA transport. In conclusion, the ESCRT regulator Did2 coordinates precise maturation of endosomes and thus provides the correct membrane identity for efficient endosomal long-distance transport