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

Microtubule-dependent mRNA transport is coupled to endosomes and important for unipolar growth in Ustilago maydis

mRNA transport is an important mechanism to orchestrate polarised growth. Although basic principles of mRNA transport are well understood, key questions still remain open: Is mRNA transport connected to membrane trafficking and why do mRNAs move bidirectionally without accumulating subcellularly? The phytopathogenic basidiomycete Ustilago maydis grows in a highly polar manner. On the surface of its host plant maize fungal hyphae migrate via extended tip growth, which is a prerequisite for infection. Recently, the RNA recognition motif-harbouring protein Rrm4 was shown to be important for this asymmetric growth, since deletion of the corresponding gene causes an increased number of hyphae to establish two growth cones. Previous in vivo studies furthermore showed that Rrm4 binds RNAs and shuttles rapidly along microtubules. In this study RNA live imaging revealed that Rrm4 mediates cytoplasmic shuttling of a set of mRNAs encoding for example the ubiquitin fusion protein Ubi1, the small G-protein Rho3 and septin Cdc3. However, the investigated mRNAs do not accumulate subcellularly. It was demonstrated that mRNA binding is essential for Rrm4 function and polar growth. Furthermore, the molecular motors kinesin-3 and dynein carry out antero- and retrograde transport of mRNAs. Intriguingly, the same motors that are mandatory for mRNA transport also mediate endosome trafficking. Investigating septin mRNA transport applying dynamic live cell imaging revealed Rrm4-mediated co-transport of septin mRNA and encoded protein. Septins are highly conserved GTPases that function either in limiting membrane diffusion or as scaffold. Septins polymerise into higher-order structures and are involved in symmetry breaking and polar growth. Since co-localisation of mRNA and protein is indicative for local protein synthesis it was tested whether septin mRNA is directly translated on endosomes. Consistently, the accumulation of septin protein on endosomes is tightly linked to the recruitment of septin mRNA. Furthermore, ribosomal proteins co-localise with shuttling endosomes, but only in the presence of mRNA. Importantly, endosomal trafficking is essential for an efficient delivery of septin protein to septin filaments at growth cones. In sum, the novel mechanism of mRNA-hitchhiking on endosomes that are trafficking due to the action of kinesin-3 and dynein was uncovered. This constitutes a novel mechanism for loading endosomes with septin protein by local translation - a process that might be important for rapid and coordinated assembly of septin filaments at hyphal growth cones

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

The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants ($\Delta$pro11, $\Delta$pro11$\Delta$pro22, $\Delta$pp2Ac1$\Delta$pro22) from the filamentous fungus $\textit {Sordaria macrospora}$. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth

Identification of Feldin, an antifungal polyyne from the beefsteak fungus Fistulina hepatica\textit {Fistulina hepatica}

Fruiting body-forming members of the Basidiomycota maintain their ecological fitness against various antagonists like ascomycetous mycoparasites. To achieve that, they produce myriads of bioactive compounds, some of which are now being used as agrochemicals or pharmaceutical lead structures. Here, we screened ethyl acetate crude extracts from cultures of thirty-five mushroom species for antifungal bioactivity, for their effect on the ascomycete $\textit {Saccharomyces cerevisiae}$ and the basidiomycete $\textit {Ustilago maydis}$. One extract that inhibited the growth of $\textit {S. cerevisiae}$ much stronger than that of $\textit {U. maydis}$ was further analyzed. For bioactive compound identification, we performed bioactivity-guided HPLC/MS fractionation. Fractions showing inhibition against $\textit {S. cerevisiae}$ but reduced activity against $\textit {U. maydis}$ were further analyzed. NMR-based structure elucidation from one such fraction revealed the polyyne we named feldin, which displays prominent antifungal bioactivity. Future studies with additional mushroom-derived eukaryotic toxic compounds or antifungals will show whether $\textit {U. maydis}$ could be used as a suitable host to shortcut an otherwise laborious production of such mushroom compounds, as could recently be shown for heterologous sesquiterpene production in $\textit {U. maydis}$