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

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

Role for RNA-Binding Proteins Implicated in Pathogenic Development of Ustilago maydis

Ustilago maydis causes smut disease on corn. Successful infection depends on a number of morphological transitions, such as pheromone-dependent formation of conjugation tubes and the switch to filamentous dikaryotic growth, as well as different types of mycelial structures during growth within the host plant. In order to address the involvement of RNA-binding proteins during this developmental program, we identified 27 open reading frames from the genome sequence encoding potential RNA-binding proteins. They exhibit similarities to RNA-binding proteins with Pumilio homology domains (PUM), the K homology domain (KHD), the double-stranded RNA binding motif (DSRM), and the RNA recognition motif (RRM). For 18 of these genes, we generated replacement mutants in compatible haploid strains. Through analysis of growth behavior, morphology, cyclic AMP response, mating, and pathogenicity, we identified three candidates with aberrant phenotypes. Loss of Khd1, a K homology protein containing three KHDs, resulted in a cold-sensitive growth phenotype. Deletion of khd4 encoding a protein with five KHDs led to abnormal cell morphology, reduced mating, and virulence. rrm4Δ strains were affected in filamentous growth and pathogenicity. Rrm4 is an RRM protein with a so far unique domain organization consisting of three N-terminal RRMs as well as a domain found in the C terminus of poly(A)-binding proteins. These results indicate a role for RNA-binding proteins in regulation of morphology as well as in pathogenic development in U. maydis

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

Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner

Abstract Background Lung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host–pathogen interactions during pneumococcal infections. Methods Following in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid. Results In vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment. Conclusion Streptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms. Video Abstrac

Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells.

The nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 are mammalian cytosolic pattern recognition receptors sensing bacterial peptidoglycan fragments in order to initiate cytokine expression and pathogen host defense. Since endothelial cells are relevant cells for pathogen recognition at the blood/tissue interface, we here analyzed the role of NOD1- and NOD2-dependently expressed microRNAs (miRNAs, miR) for cytokine regulation in murine pulmonary endothelial cells. The induction of inflammatory cytokines in response to NOD1 and NOD2 was confirmed by increased expression of tumour necrosis factor (Tnf)-α and interleukin (Il)-6. MiRNA expression profiling revealed NOD1- and NOD2-dependently regulated miRNA candidates, of which miR-147-3p, miR-200a-3p, and miR-298-5p were subsequently validated in pulmonary endothelial cells isolated from Nod1/2-deficient mice. Analysis of the two down-regulated candidates miR-147-3p and miR-298-5p revealed predicted binding sites in the 3' untranslated region (UTR) of the murine Tnf-α and Il-6 mRNA. Consequently, transfection of endothelial cells with miRNA mimics decreased Tnf-α and Il-6 mRNA levels. Finally, a novel direct interaction of miR-298-5p with the 3' UTR of the Il-6 mRNA was uncovered by luciferase reporter assays. We here identified a mechanism of miRNA-down-regulation by NOD stimulation thereby enabling the induction of inflammatory gene expression in endothelial cells