CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.

Das Zytosol ist der Hauptort der Proteinbiosynthese. Während viele Proteine im Zytosol bleiben, muss ein Großteil zu unterschiedlichen Kompartimenten der Zelle transportiert werden. Die korrekte Lokalisation der Polypeptide ist essentiell für die Homöostase der Zelle. Werden Proteine fehlgeleitet oder gar nicht transportiert, können diese in der Zelle aggregieren, was zu Stress bis hin zum Zelltod führen kann. Obwohl der Import mitochondrialer Proteine über die verschiedenen Membranen der Mitochondrien sehr gut erforscht ist, war lange unklar, wie diese Proteine zu ihrem Zielorganell transportiert werden. In den letzten Jahren wurde diese Wissenslücke teilweise gefüllt, neue zytosolische Faktoren wurden identifiziert und alternative Transportwege aufgedeckt. Eine solche Entdeckung war der Transportweg namens ER-SURF. Hier werden mitochondriale Proteine an die Membran des endoplasmatischen Retikulums transportiert, wo sie vom Co-Chaperon Djp1 gebunden und zu den Mitochondrien gebracht werden. Im Zuge der Studie zu ER-SURF wurde ein Protein identifiziert, das bisher noch uncharakterisiert war. Dieses Protein nannten wir Ema19 („Efficient Mitochondria Targeting–Associated Protein 19”). Es ist ein Membranprotein des endoplasmatischen Retikulums, das vier Transmembrandomänen besitzt. Ziel dieses Projekts war es, die Funktion von Ema19 für die Zelle zu analysieren. Durch ein Alignment konnte ich feststellen, dass das Protein bis in den Menschen hoch konserviert ist, was auf eine wichtige Rolle für die Zelle schließen ließ. Da Ema19 im Zusammenhang mit dem ER-SURF Transportweg identifiziert wurde, habe ich zunächst eine mögliche Rolle für den Transport und Import mitochondrialer Proteine in unterschiedlichen Experimenten getestet. Im Laufe der Arbeit wurde jedoch deutlich, dass Ema19 keine direkte Rolle beim Import von mitochondrialen Proteinen spielt. Allerdings konnte ich durch mehrere unabhängige Versuche einen Zusammenhang mit der Lokalisation und dem Abbau mitochondrialer Proteine feststellen. Fehlt Ema19 in der Zelle, ist vor allem das mitochondriale Protein Oxa1 mehr am endoplasmatischen Retikulum vorzufinden. Ebenso konnte ich feststellen, dass Oxa1, sowie das Intermembranraumprotein Erv1, langsamer abgebaut werden als in Wildtypzellen. Diese Experimente geben erste Hinweise auf eine mögliche Rolle von Ema19 für den Abbau mitochondrialer Proteine an der ER-Membran. Nichtsdestotrotz bleiben noch viele Fragen offen und weitere Versuche sind nötig, um diese Hypothese weiter zu unterstützen.

The number of sequenced genomes increases rapidly due to the development of faster, better and new technologies. Thus, there is a great interest in automation, and standardization of the subsequent processing and analysis stages of the generated enormous amount of data. In the current work, genomes of clones, strains and species of Streptococcus were compared, which were sequenced, annotated and analysed with several technologies and methods. For sequencing, the 454- and Illumina-technology were used. The assembly of the genomes mainly was performed by the gsAssembler (Newbler) of Roche, the annotation was performed by the annotation pipeline RAST, the transfer tool RATT or manually. Concerning analysis, sets of deduced proteins of several genomes were compared to each other and common components, the so-called core-genome, of the used genomes of one or closely related species determined. Detailed comparative analysis was performed for the genomes of isolates of two clones to gather single nucleotide variants (SNV) within genes. This work focusses on the pathogenic organism Streptococcus pneumoniae. This species is a paradigm for transformability, virulence and pathogenicity as well as resistance mechanisms against antibiotics. Its close relatives S. mitis, S. pseudopneumoniae and S. oralis have no pathogenicity potential as high as S. pneumoniae available and are thus of high interest to understand the evolution of S. pneumoniae. Strains of two S. pneumoniae clones were chosen. One is the ST10523 clone, which is associated with patients with cystic fibrosis and is characterized by long-term persistence. This clone is lacking an active hyaluronidase, which is one of the main virulence factors. The lack of two phage clusters possibly contributed to the long persistence in the human host. The clone ST226 shows a high penicillin resistance but interestingly one strain is sensitive against penicillin. Here it could be seen that the penicillin resistance mainly arose from the presence of mosaic-PBPs, while special alleles of MurM and CiaH - both genes are associated with penicillin-resistance – were present in resistant and sensitive strains as well. Penicillin resistance of S. pneumoniae is the result of horizontal gene transfer, where DNA of closely related species, mainly S. mitis or S. oralis, served as donor. The transfer of DNA from the high-level penicillin-resistant strain S. oralis Uo5 to the sensitive strain S. pneumoniae R6 was intentioned to reveal the amount of transferred DNA and whether it is possible to reach the high resistance level of S. oralis Uo5. Altogether, about 19kb of S. oralis DNA were transferred after three successive transformation steps, about 10-fold less than during transfer from S. mitis, which is more closely related to S. pneumoniae, as donor. MurE was identified as new resistance determinant. Since the resistance level of the donor strain could not be reached, it is assumed, that further unknown factors are present which contribute to penicillin resistance. The comparison of S. pneumoniae and its close relatives was performed using deduced protein sequences. 1.041 homologous proteins are common to the four complete genomes of S. pneumoniae R6, S. pseudopneumoniae IS7493, S. mitis B6 and S. oralis Uo5. Most of the virulence and pathogenicity factors described for S. pneumoniae could also be found in commensal species. These observations were confirmed by further investigations by Kilian et al. (Kilian, et al., 2019). After adding 26 complete S. pneumoniae genomes to the analysis, only 104 gene products could be identified as specific for this species. Investigations of a larger number of related streptococci, which were isolated from human and several primates, confirmed the presence of most of the virulence factors of human pneumococci in S. oralis and S. mitis strains from primates. While NanBC is common among S. pneumoniae and is missing in all S. oralis, all S. oralis contain a ß-N-acetyl-hexosaminidase which vice versa is missing in S. pneumoniae. The occurrence of S. oralis also in free-living chimpanzees suggests the assumption, that this species is part of the commensal flora of these Old-World monkeys unlike S. pneumoniae which has evolved with its human host. Compared to S. pneumoniae, S. oralis shows an amazing variability in factors important for biosynthesis of peptidoglycan and teichoic acid (PBP, MurMN, lic-cluster). Some streptococci contain a second PGP3 homologue. Additional analyses with further isolates, especially of wild animals, are necessary to determine host-specific components.

Cell division and cell elongation are fundamental processes for growth. In contrast to animal cells, plant cells are surrounded by rigid walls and therefore loosening of the wall is required during elongation. On the other hand, vacuole size has been shown to correlate with cell size and inhibition of vacuolar expansion limits cell growth. However, the specific role of the vacuole during cell elongation is still not fully resolved. Especially the question whether the vacuole is the leading unit during cellular growth or just passively expands upon water uptake remains to be answered. Here, we review recent findings about the contribution of the vacuole to cell elongation. In addition, we also discuss the connection between cell wall status and vacuolar morphology. In particular, we focus on the question whether vacuolar size is dictated by cell size or vice versa and share our personnel view about the sequential steps during cell elongation.

Cells depend on the continuous renewal of their proteome composition during the cell cycle and in order to replace aberrant proteins or to react to changing environmental conditions. In higher eukaryotes, protein synthesis is achieved by up to five million ribosomes per cell. With the fast kinetics of translation, the large number of newly made proteins generates a substantial burden for protein homeostasis and requires a highly orchestrated cascade of factors promoting folding, sorting and final maturation. Several of the involved factors directly bind to translating ribosomes for the early processing of emerging nascent polypeptides and the translocation of ribosome nascent chain complexes to target membranes. In plant cells, protein synthesis also occurs in chloroplasts serving the expression of a relatively small set of 60–100 protein-coding genes. However, most of these proteins, together with nucleus-derived subunits, form central complexes majorly involved in the essential processes of photosynthetic light reaction, carbon fixation, metabolism and gene expression. Biogenesis of these heterogenic complexes adds an additional level of complexity for protein biogenesis. In this review, we summarize the current knowledge about co-translationally binding factors in chloroplasts and discuss their role in protein folding and ribosome translocation to thylakoid membranes.

Cancer cells often harbor chromosomes in abnormal numbers and with aberrant structure. The consequences of these chromosomal aberrations are difficult to study in cancer, and therefore several model systems have been developed in recent years. We show that human cells with extra chromosome engineered via microcell-mediated chromosome transfer often gain massive chromosomal rearrangements. The rearrangements arose by chromosome shattering and rejoining as well as by replication-dependent mechanisms. We show that the isolated micronuclei lack functional lamin B1 and become prone to envelope rupture, which leads to DNA damage and aberrant replication. The presence of functional lamin B1 partly correlates with micronuclei size, suggesting that the proper assembly of nuclear envelope might be sensitive to membrane curvature. The chromosomal rearrangements in trisomic cells provide growth advantage compared to cells without rearrangements. Our model system enables to study mechanisms of massive chromosomal rearrangements of any chromosome and their consequences in human cells.

The Atacama Desert is one of the driest and probably oldest deserts on Earth where only a few extremophile organisms are able to survive. This study investigated two terricolous and two epiphytic lichens from the fog oasis “Las Lomitas” within the National Park Pan de Azúcar which represents a refugium for a few vascular desert plants and many lichens that can thrive on fog and dew alone. Ecophysiological measurements and climate records were combined with molecular data of the mycobiont, their green algal photobionts and lichenicolous fungi to gain information about the ecology of lichens within the fog oasis. Phylogenetic and morphological investigations led to the identification and description of the new lichen species Acarospora conafii sp. nov. as well as the lichenicolous fungi that accompanied them and revealed the trebouxioid character of all lichen photobionts. Their photosynthetic responses were compared during natural scenarios such as reactivation by high air humidity and in situ fog events to elucidate the activation strategies of this lichen community. Epiphytic lichens showed photosynthetic activity that was rapidly induced by fog and high relative air humidity whereas terricolous lichens were only activated by fog.

Biological clocks exist across all life forms and serve to coordinate organismal physiology with periodic environmental changes. The underlying mechanism of these clocks is predominantly based on cellular transcription-translation feedback loops in which clock proteins mediate the periodic expression of numerous genes. However, recent studies point to the existence of a conserved timekeeping mechanism independent of cellular transcription and translation, but based on cellular metabolism. These metabolic clocks were concluded based upon the observation of circadian and ultradian oscillations in the level of hyperoxidized peroxiredoxin proteins. Peroxiredoxins are enzymes found almost ubiquitously throughout life. Originally identified as H2O2 scavengers, recent studies show that peroxiredoxins can transfer oxidation to, and thereby regulate, a wide range of cellular proteins. Thus, it is conceivable that peroxiredoxins, using H2O2 as the primary signaling molecule, have the potential to integrate and coordinate much of cellular physiology and behavior with metabolic changes. Nonetheless, it remained unclear if peroxiredoxins are passive reporters of metabolic clock activity or active determinants of cellular timekeeping. Budding yeast possess an ultradian metabolic clock termed the Yeast Metabolic Cycle (YMC). The most obvious feature of the YMC is a high amplitude oscillation in oxygen consumption. Like circadian clocks, the YMC temporally compartmentalizes cellular processes (e.g. metabolism) and coordinates cellular programs such as gene expression and cell division. The YMC also exhibits oscillations in the level of hyperoxidized peroxiredoxin proteins. In this study, I used the YMC clock model to investigate the role of peroxiredoxins in cellular timekeeping, as well as the coordination of cell division with the metabolic clock. I observed that cytosolic 2-Cys peroxiredoxins are essential for robust metabolic clock function. I provide direct evidence for oscillations in cytosolic H2O2 levels, as well as cyclical changes in oxidation state of a peroxiredoxin and a model peroxiredoxin target protein during the YMC. I noted two distinct metabolic states during the YMC: low oxygen consumption (LOC) and high oxygen consumption (HOC). I demonstrate that thiol-disulfide oxidation and reduction are necessary for switching between LOC and HOC. Specifically, a thiol reductant promotes switching to HOC, whilst a thiol oxidant prevents switching to HOC, forcing cells to remain in LOC. Transient peroxiredoxin inactivation triggered rapid and premature switching from LOC to HOC. Furthermore, I show that cell division is normally synchronized with the YMC and that deletion of typical 2-Cys peroxiredoxins leads to complete uncoupling of cell division from metabolic cycling. Moreover, metabolic oscillations are crucial for regulating cell cycle entry and exit. Intriguingly, switching to HOC is crucial for initiating cell cycle entry whilst switching to LOC is crucial for cell cycle completion and exit. Consequently, forcing cells to remain in HOC by application of a thiol reductant leads to multiple rounds of cell cycle entry despite failure to complete the preceding cell cycle. On the other hand, forcing cells to remain in LOC by treating with a thiol oxidant prevents initiation of cell cycle entry. In conclusion, I propose that peroxiredoxins – by controlling metabolic cycles, which are in turn crucial for regulating the progression through cell cycle – play a central role in the coordination of cellular metabolism with cell division. This proposition, thus, positions peroxiredoxins as active players in the cellular timekeeping mechanism.

In cyanobacteria and plants, VIPP1 plays crucial roles in the biogenesis and repair of thylakoid membrane protein complexes and in coping with chloroplast membrane stress. In chloroplasts, VIPP1 localizes in distinct patterns at or close to envelope and thylakoid membranes. In vitro, VIPP1 forms higher-order oligomers of >1 MDa that organize into rings and rods. However, it remains unknown how VIPP1 oligomerization is related to function. Using time-resolved fluorescence anisotropy and sucrose density gradient centrifugation, we show here that Chlamydomonas reinhardtii VIPP1 binds strongly to liposomal membranes containing phosphatidylinositol-4-phosphate (PI4P). Cryo-electron tomography reveals that VIPP1 oligomerizes into rods that can engulf liposomal membranes containing PI4P. These findings place VIPP1 into a group of membrane-shaping proteins including epsin and BAR domain proteins. Moreover, they point to a potential role of phosphatidylinositols in directing the shaping of chloroplast membranes.

The structural integrity of synaptic connections critically depends on the interaction between synaptic cell adhesion molecules (CAMs) and the underlying actin and microtubule cytoskeleton. This interaction is mediated by giant Ankyrins, that act as specialized adaptors to establish and maintain axonal and synaptic compartments. In Drosophila, two giant isoforms of Ankyrin2 (Ank2) control synapse stability and organization at the larval neuromuscular junction (NMJ). Both Ank2-L and Ank2-XL are highly abundant in motoneuron axons and within the presynaptic terminal, where they control synaptic CAMs distribution and organization of microtubules. Here, we address the role of the conserved N-terminal ankyrin repeat domain (ARD) for subcellular localization and function of these giant Ankyrins in vivo. We used a P[acman] based rescue approach to generate deletions of ARD subdomains, that contain putative binding sites of interacting transmembrane proteins. We show that specific subdomains control synaptic but not axonal localization of Ank2-L. These domains contain binding sites to L1-family member CAMs, and we demonstrate that these regions are necessary for the organization of synaptic CAMs and for the control of synaptic stability. In contrast, presynaptic Ank2-XL localization only partially depends on the ARD but strictly requires the presynaptic presence of Ank2-L demonstrating a critical co-dependence of the two isoforms at the NMJ. Ank2-XL dependent control of microtubule organization correlates with presynaptic abundance of the protein and is thus only partially affected by ARD deletions. Together, our data provides novel insights into the synaptic targeting of giant Ankyrins with relevance for the control of synaptic plasticity and maintenance.